Hemoglobin alpha chain peptide fragments useful for inhibiting stem cell proliferation

ABSTRACT

Disclosed and claimed are methods for the isolation and use of stem cell inhibiting factors for regulating the abnormal stem cell cycle and for accelerating the post-chemotherapy peripheral blood cell recovery. Also disclosed and claimed are the inhibitors of stem cell proliferation.

This application is a continuation-in-part application of U.S. Ser. No.08/316,424 filed Sep. 30, 1994 which is a continuation-in-partapplication of U.S. Ser. No. 08/040924 filed Mar. 31, 1993, now U.S.Pat. No. 5,402,475, each of which is hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to the use of inhibitors of stem cellproliferation for regulating stem cell cycle in the treatment of humansor animals having autoimmune diseases, aging, cancer, myelodysplasia,preleukemia, leukemia, psoriasis or other diseases involvinghyperproliferative conditions. The present invention also relates to amethod of treatment for humans or animals anticipating or havingundergone exposure to chemotherapeutic agents, other agents which damagecycling stem cells, or radiation exposure. Finally, the presentinvention relates to the improvement of the stem cell maintenance orexpansion cultures for auto- and allo-transplantation procedures or forgene transfer.

BACKGROUND OF THE INVENTION

Most end-stage cells in renewing systems are short-lived and must bereplaced continuously throughout life. For example, blood cellsoriginate from a self-renewing population of multipotent hematopoieticstem cells (HSC). Hematopoietic stem cells are a subpopulation ofhematopoietic cells. Hematopoietic cells can be obtained, for example,from bone marrow, umbilical cord blood or peripheral blood (eitherunmobilized or mobilized with an agent such as G-CSF); hematopoieticcells include the stem cell population, progenitor cells, differentiatedcells, accessory cells, stromal cells and other cells that contribute tothe environment necessary for production of mature blood cells. Becausethe hematopoietic stem cells are necessary for the development of all ofthe mature cells of the hematopoietic and immune systems, their survivalis essential in order to reestablish a fully functional host defensesystem in subjects treated with chemotherapy or other agents.

Hematopoietic cell production is regulated by a series of factors thatstimulate growth and differentiation of hematopoietic cells, some ofwhich, for example erythropoietin and G-CSF, are currently used inclinical practice. One part of the control network which has not beenextensively characterized, however, is the feedback mechanism that formsthe negative arm of the regulatory process (Eaves et al. Blood78:110-117, 1991).

Early studies by Lord and coworkers showed the existence of a solubleprotein factor in normal murine and porcine bone marrow extracts, whichwas capable of reversibly inhibiting the cycling of HSC (Lord et al.,Br. J. Haem. 34:441-446, 1976). This inhibitory activity (50-100 kDmolecular weight) was designated stem cell inhibitor (SCI).

Purification of this factor from primary sources was not accomplisheddue to the difficulties inherent in an in vivo assay requiring largenumbers of irradiated mice. In an attempt to overcome these problemsPragnell and co-workers developed an in vitro assay for primitivehematopoietic cells (CFU-A) and screened cell lines as a source of theinhibitory activity (see Graham et al. Nature 344:442-444, 1990).

As earlier studies had identified macrophages as possible sources forSCI (Lord et al. Blood Cells 6:581-593, 1980), a mouse macrophage cellline, J774.2, was selected (Graham et al. Nature 344:442-444, 1990). Theconditioned medium from this cell line was used by Graham et al. forpurification; an inhibitory peptide was isolated which proved to beidentical to the previously described cytokine macrophage inflammatoryprotein 1-alpha (MIP-1α). Thus, MIP-1α was isolated from a cell line,not from primary material. While Graham et al. observed that antibody toMIP-1α abrogated the activity of a crude bone marrow extract, otherworkers have shown that other inhibitory activities are important. Forexample, Graham et al. (J. Exp. Med. 178:925-32, 1993) have suggestedthat TGFβ, not MIP-1α, is a primary inhibitor of hematopoietic stemcells. Further, Eaves et al. (PNAS 90:12015-19, 1993) have suggestedthat both MIP-1α and TGFβ are present at sub optimal levels in normalbone marrow and that inhibition requires a synergy between the twofactors.

Other workers have described additional stem cell inhibitory factors.Frindel and coworkers have isolated a tetrapeptide from fetal calfmarrow and from liver extracts which has stem cell inhibitory activities(Lenfant et al., PNAS 86:779-782, 1989). Paukovits et al. (Cancer Res.50:328-332, 1990) have characterized a pentapeptide which, in itsmonomeric form, is an inhibitor and, in its dimeric form, is astimulator of stem cell cycling. Other factors have also been claimed tobe inhibitory in various in vitro systems (see Wright and Pragnell inBailliere's Clinical Haematology v. 5, pp. 723-39, 1992 (BailliereTinadall, Paris)).

Tsyrlova et al., SU 1561261 A1, disclosed a purification process for astem cell proliferation inhibitor.

To date, none of these factors have been approved for clinical use.However, the need exists for effective stem cell inhibitors. The majortoxicity associated with chemotherapy or radiation treatment is thedestruction of normal proliferating cells which can result in bonemarrow suppression or gastrointestinal toxicity. An effective stem cellinhibitor would protect these cells and allow for the optimization ofthese therapeutic regimens. Just as there is a proven need for a varietyof stimulatory cytokines (i.e., cytokines such as IL-1, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, IL-13, IL-14, IL-15, G-CSF, GM-CSF,erythropoietin, thrombopoietin, stem cell factor, flk2/flt3 ligand,etc., which stimulate the cycling of hematopoietic cells ) dependingupon the clinical situation, so too it is likely that a variety ofinhibitory factors will be needed to address divergent clinical needs.

Hemoglobin is a highly conserved tetrameric protein with molecularweight of approximately 64,000 Daltons. It consists of two alpha and twobeta chains. Each chain binds a single molecule of heme(ferroprotoporphyrin IX), an iron-containing prosthetic group.Vertebrate alpha and beta chains were probably derived from a singleancestral gene which duplicated and then diverged; the two chains retaina large degree of sequence identity both between themselves and betweenvarious vertebrates (see FIG. 16A). In humans, the alpha chain clusteron chromosome 16 contains two alpha genes (alpha₁ and alpha₂) which codefor identical polypeptides, as well as genes coding for other alpha-likechains: zeta, theta and several non-transcribed pseudogenes (see FIG.16B for cDNA and amino acid sequences of human alpha chain). The betachain cluster on chromosome 11 consists of one beta chain gene andseveral beta-like genes: delta, epsilon, G gamma and A gamma, as well asat least two unexpressed pseudogenes (see FIG. 16C for cDNA and aminoacid sequences of human beta chain).

The expression of these genes varies during development. In humanhematopoiesis, which has been extensively characterized, embryonicerythroblasts successively synthesize tetramers of two zeta chains andtwo epsilon chains (Gower I), two alpha chains and two epsilon chains(Gower II) or two zeta chains and two gamma chains (Hb Portland). Asembryogenesis proceeds, the predominant form consists of fetalhemoglobin (Hb F) which is composed of two alpha chains and two gammachains. Adult hemoglobin (two alpha and two beta chains) begins to besynthesized during the fetal period; at birth approximately 50% ofhemoglobin is of the adult form and the transition is complete by about6 months of age. The vast majority of hemoglobin (approximately 97%) inthe adult is of the two alpha and two beta chain variety (Hb A) withsmall amounts of Hb F or of delta chain (Hb A₂) being detectable.

Heme has been extensively examined with regard to its influences onhematopoiesis (see S. Sassa, Seminars Hemat. 25:312-20, 1988 and N.Abraham et al., Int. J. Cell Cloning 9:185-210, 1991 for reviews). Hemeis required for the maturation of erythroblasts; in vitro, hemin(chloroferroprotoporphyrin IX - i.e., heme with an additional chlorideion) increases the proliferation of CFU-GEMM, BFU-E and CFU-E.Similarly, hemin increases cellularity in long-term bone marrowcultures.

I. Chemotherapy and Radiotherapy of Cancer

Productive research on stimulatory growth factors has resulted in theclinical use of a number of these factors (erythropoietin, G-CSF,GM-CSF, etc.). These factors have reduced the mortality and morbidityassociated with chemotherapeutic and radiation treatments. Furtherclinical benefits to patients who are undergoing chemotherapy orradiation could be realized by an alternative strategy of blockingentrance of stem cells into cell cycle thereby protecting them fromtoxic side effects.

II. Bone Marrow Transplantation

Bone marrow transplantation (BMT) is a useful treatment for a variety ofhematological, autoimmune and malignant diseases; current therapiesinclude hematopoietic cells obtained from umbilical cord blood or fromperipheral blood (either unmobilized or mobilized with agents such asG-CSF) as well as from bone marrow. Ex vivo manipulation ofhematopoietic cells is currently being used to expand primitive stemcells to a population suitable for transplantation. Optimization of thisprocedure requires: (1) sufficient numbers of stem cells able tomaintain long term reconstitution of hematopoiesis; (2) the depletion ofgraft versus host-inducing T-lymphocytes and (3) the absence of residualmalignant cells. This procedure can be optimized by including a stemcell inhibitor(s) for ex vivo expansion.

The effectiveness of purging of hematopoietic cells with cytotoxic drugsin order to eliminate residual malignant cells is limited due to thetoxicity of these compounds for normal hematopoietic cells andespecially stem cells. There is a need for effective protection ofnormal cells during purging; protection can be afforded by taking stemcells out of cycle with an effective inhibitor.

III. Peripheral Stem Cell Harvesting

Peripheral blood stem cells (PBSC) offer a number of potentialadvantages over bone marrow for autologous transplantation. Patientswithout suitable marrow harvest sites due to tumor involvement orprevious radiotherapy can still undergo PBSC collections. The use ofblood stem cells eliminates the need for general anesthesia and asurgical procedure in patients who would not tolerate this well. Theapheresis technology necessary to collect blood cells is efficient andwidely available at most major medical centers. The major limitations ofthe method are both the low normal steady state frequency of stem cellsin peripheral blood and their high cycle status after mobilizationprocedures with drugs or growth factors (e.g., cyclophosphamide, G-CSF,stem cell factor). An effective stem cell inhibitor would be useful toreturn such cells to a quiescent state, thereby preventing their lossthrough differentiation.

IV. Treatment of Hyperproliferative Disorders

A number of diseases are characterized by a hyperproliferative state inwhich dysregulated stem cells give rise to an overproduction of endstage cells. Such disease states include, but are not restricted to,psoriasis, in which there is an overproduction of epidermal cells, andpremalignant conditions in the gastrointestinal tract characterized bythe appearance of intestinal polyps. A stem cell inhibitor would beuseful in the treatment of such conditions.

V. Gene Transfer

The ability to transfer genetic information into hematopoietic cells iscurrently being utilized in clinical settings. Hematopoietic cells are auseful target for gene therapy because of ease of access, extensiveexperience in manipulating and treating this tissue ex vivo and becauseof the ability of blood cells to permeate tissues. Furthermore, thecorrection of certain human genetic defects may be possible by theinsertion of a functional gene into the primitive stem cells of thehuman hematopoietic system.

There are several limitations for the introduction of genes into humanhematopoietic cells using either retrovirus vectors or physicaltechniques of gene transfer: (1) The low frequency of stem cells inhematopoietic tissues has necessitated the development of highefficiency gene transfer techniques; and (2) more rapidly cycling stemcells proved to be more susceptible to vector infection, but theincrease of the infection frequency by stimulation of stem cellproliferation with growth factors produces negative effects on long termgene expression, because cells containing the transgenes are forced todifferentiate irreversibly and lose their self-renewal. These problemscan be ameliorated by the use of a stem cell inhibitor to preventdifferentiation and loss of self-renewal.

SUMMARY OF THE INVENTION

The present invention relates to polypeptides which are inhibitors ofstem cell proliferation ("INPROL") and their use.

The present invention includes an inhibitor of stem cell proliferationcharacterized by the following properties:

(a) Specific activity (IC₅₀) less than or equal to 20 ng/ml in a murinecolony-forming spleen (CFU-S) assay (see Example 4),

(b) Molecular weight greater than 10,000 and less than 100,000 daltons(by ultrafiltration),

(c) Activity sensitive to degradation by trypsin,

(d) More hydrophobic than MIP-1αor TGFβ and separable from both byreverse phase chromatography (see Example 12),

(e) Biological activity retained after heating for one hour at 37° C.,55° C. or 75° C. in aqueous solution and

(f) Biological activity retained after precipitation with 1%hydrochloric acid in acetone.

The present invention is further characterized and distinguished fromother candidate stem cell inhibitors (e.g., MIP-1α, TGFβ band variousoligopeptides) by its capacity to achieve inhibition in an in vitroassay after a short preincubation period (see Example 5).

The present invention also comprises pharmaceutical compositionscontaining INPROL for treatment of a variety of disorders.

The present invention provides a method of treating a subjectanticipating exposure to an agent capable of killing or damaging stemcells by administering to that subject an effective amount of a stemcell inhibitory composition. The stem cells protected by this method maybe hematopoietic stem cells ordinarily present and dividing in the bonemarrow. Alternatively, stem cells may be epithelial, located forexample, in the intestines or scalp or other areas of the body or germcells located in reproductive organs. The method of this invention maybe desirably employed on humans, although animal treatment is alsoencompassed by this method. As used herein, the terms "subject" or"patient" refer to an animal, such as a mammal, including a human.

In another aspect, the invention provides a method for protecting andrestoring the hematopoietic, immune or other stem cell systems of apatient undergoing chemotherapy, which includes administering to thepatient an effective amount of INPROL.

In still a further aspect, the present invention involves a method foradjunctively treating any cancer, including those characterized by solidtumors, by administering to a patient having cancer an effective amountof INPROL to protect stem cells of the bone marrow, gastrointestinaltract or other organs from the toxic effects of chemotherapy orradiation therapy.

Yet another aspect of the present invention involves the treatment ofleukemia, comprising treating hematopoietic cells having proliferatingleukemia cells therein with an effective amount of INPROL to inhibitproliferation of normal stem cells, and treating the bone marrow with acytotoxic agent to destroy leukemia cells. This method may be enhancedby the follow-up treatment of the bone marrow with other agents thatstimulate its proliferation; e.g., colony stimulating factors. In oneembodiment this method is performed in vivo. Alternatively, this methodis also useful for ex vivo purging and expansion of hematopoietic cellsfor transplantation.

In still a further aspect, the method involves treating a subject havingany disorder caused by proliferating stem cells. Such disorders, such aspsoriasis, myelodysplasia, some autoimmune diseases, immuno-depressionin aging, are treated by administering to the subject an effectiveamount of INPROL to partially inhibit proliferation of the stem cell inquestion.

The present invention provides a method for reversibly protecting stemcells from damage from a cytotoxic agent capable of killing or damagingstem cells. The method involves administering to a subject anticipatingexposure to such an agent an effective amount of INPROL.

The present invention also provides:

An inhibitor of stem cell proliferation isolated from porcine or otherbone marrow by the following procedure (see Example 12):

(a) Extraction of bone marrow and removal of particulate matter throughfiltration,

(b) Heat treatment at 56° C. for 40 minutes followed by cooling in icebath,

(c) Removal of precipitate by centrifugation at 10,000 g for 30 minutesat 4° C.,

(d) Acid precipitation by addition of supernatant to 10 volumes ofstirred ice-cold acetone containing 1% by volume concentratedhydrochloric acid and incubation at 4° C. for 16 hours,

(e) Isolation of precipitate by centrifugation at 20,000 g for 30minutes at 4° C. and washing with cold acetone followed by drying,

(f) Isolation by reverse phase chromatography and monitoring activity byinhibition of colony formation by bone marrow cells pretreated with5-fluorouracil and incubated in the presence of murine IL-3, as well asby absorption at 280 nm and by SDS-PAGE.

The present invention also provides:

A method for purifying an inhibitor of stem cell proliferationsubstantially free from other proteinaceous materials comprising thepreceding steps, as also described in more detail below.

The present invention also provides:

A method of treatment for humans or animals wherein an inhibitor of stemcell proliferation functions to ameliorate immunosuppression caused bystem cell hyperproliferation.

The present invention also provides:

A method of treatment for humans or animals wherein said inhibitor ofstem cell proliferation is administered after the stem cells are inducedto proliferate by exposure to a cytotoxic drug or irradiation procedure.Stem cells are normally quiescent but are stimulated to enter cell cycleafter chemotherapy. This renders them more sensitive to a secondadministration of chemotherapy; the current method protects them fromthis treatment.

The present invention also provides:

A method of treatment for humans or animals wherein said inhibitor ofstem cell proliferation is administered as an adjuvant before ortogether with vaccination for the purpose of increasing immune response.

The present invention also provides:

A method of treatment for humans or animals receiving cytotoxic drugs orradiation treatment which comprises administering an effective amount ofthe inhibitor of stem cell proliferation to protect stem cells againstdamage.

The invention also includes a pharmaceutical composition comprisinghemoglobin and a pharmaceutically acceptible carrier.

The invention also includes a pharmaceutical composition comprising (a)a polypeptide selected from the group consisting of the alpha chain ofhemoglobin, the beta chain of hemoglobin, the gamma chain of hemoglobin,the delta chain of hemoglobin, the epsilon chain of hemoglobin and thezeta chain of hemoglobin, and (b) a pharmaceutically acceptible carrier.Such pharmaceutical compositions can composed of a single polypeptideselected from said group, a mixture of polypeptides selected from saidgroup or polypeptides from said group in the form of dimers ormultimers, with or without heme.

The invention also includes peptides having the sequences:

Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala-Gln-Val (SEQ ID NO:1),

Cys-Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala-Gln-Val-Cys (SEQ IDNO:2)

where the two Cys residues form a disulfide bond,

Cys-Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala-Gln-Val-Cys

where the two Cys residues are joined by a carbon bridge, and

Asp-Ala-Leu-Thr-Asn-Ala-Val-Ala-His-Val-Asp-Asp-Met-Pro-Asn-Ala-Leu-Ser-Ala(SEQ ID NO:3).

Also included in the invention are DNA sequences encoding the aboveidentified peptides, vectors containing said DNA sequences and hostcells containing said vectors. These peptides can be synthesized usingstandard chemical techniques (e.g., solid phase synthesis) or by usingrecombinant techniques (e.g., fusion systems such as those employingglutathione-S-transferase (D. B. Smith and K. S. Johnson, Gene 67:31-40,1988), thioredoxin (LaVallie et al., Biotechnology 11:187-193, 1993) orubiquitin (Butt et al., PNAS 86:2540-4, 1989; U.S. Pat. No. 5,391,490)).

Additionally the invention includes a method of inhibiting stem cellproliferation comprising contacting hematopoietic cells with a compoundcapable of binding opiate receptors, advantageously the mu subclass ofopiate receptors. Peptides (called "hemorphins") have been isolated fromhemoglobin which exhibit opiate-like activities (e.g., Brantl et al.,Eur. J. Pharm, 125:309-10, 1986; Davis et al. Peptides 10:747-51, 1989;Karelin et al. Bioch. Biophys. Res. Comm, 202:410-5, 1994; Zhao et al.,Ann. N.Y. Acad. Sci 750:452-8, 1995). Each of these articles is herebyincorporated by reference.

The invention also includes a method of inhibiting stem cellproliferation comprising contacting hematopoietic cells with a peptideselected from the group of hemorphin peptides having the sequence:

Leu-Val-Val-Tyr-Pro-Trp-Thr-Gin-Arg-Phe, (SEQ ID NO:4)

Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg, (SEQ ID NO:5)

Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln, (SEQ ID NO:6)

Leu-Val-Val-Tyr-Pro-Trp-Thr, (SEQ ID NO:7)

Leu-Val-Val-Tyr-Pro-Trp, (SEQ ID NO:8)

Leu-Val-Val-Tyr-Pro, (SEQ ID NO:9)

Val-Val-Tyr-Pro-Trp-Thr-Gln, (SEQ ID NO:10)

Tyr-Pro-Trp-Thr-Gln-Arg-Phe, (SEQ ID NO:11)

Tyr-Pro-Trp-Thr-Gln-Arg, (SEQ ID NO:12)

Tyr-Pro-Trp-Thr-Gln, and (SEQ ID NO:13)

Tyr-Pro-Trp-Thr.

The above peptides have sequence similarity to other opiate-likepeptides such as those of the Tyr-MIF-1 family (see Reed et al.,Neurosci. Biobehav. Rev. 18:519-25, 1994 for review), the casein-derivedcasomorphins (Brantl et al., Hoppe-Seyler's Z. Physiol. Chem.360:1211-16, 1979; Loukas et al., Biochem. 22:4567-4573, 1983; Fiat andJolles, Mol. Cell. Biochem. 87:5-30, 1989), peptides derived fromcytochrome b, termed cytochrophins (Brantl et al., Eur. J. Pharm.111:293-4, 1985) as well as peptides derived from combinatoriallibraries screened for binding to opiate receptors (see Dooley et al.,Peptide Research 8:124-137, 1995 for review). Each of these articles ishereby incorporated by reference.

The invention also includes a method of inhibiting stem cellproliferation comprising contacting hematopoietic cells with a peptideselected from the group consisting of Tyr-MIF-1 related peptides,casomorphins and cytochrophins. Specifically included are the Tyr-MIF-1peptides having the sequences:

Tyr-Pro-Try-Gly-NH₂,

Tyr-Pro-Lys-Gly-NH₂,

Tyr-Pro-Leu-Gly-NH₂, and

Pro-Leu-Gly-NH₂.

The invention also includes a method of conducting gene therapy in amammal comprising:

a) removing hematopoietic cells from said mammal,

b) optionally treating said hematopoietic cells ex vivo with at leastone stimulatory cytokine to induce stem cell proliferation,

c) transfecting said hematopoietic cells with a pre-determined gene,

d) contacting said transfected hematopoietic cells ex vivo with INPROL,

e) transplanting into a mammal the hematopoietic cells of step d,

f) optionally treating said mammal in vivo with INPROL.

The invention also includes a method of conducting ex vivo stem cellexpansion comprising treating said hematopoietic cells with INPROL andat least one stimulatory cytokine. INPROL is contacted with thehematopoietic cells before, during and/or after contact with thestimulatory cytokine.

The invention also includes a pharmaceutical composition comprising (a)INPROL and (b) at least one inhibitory compound selected from the groupconsisting of MIP-1α, TGFβ, TNFα, INFα, INFβ, INFγ, the pentapeptidepyroGlu-Glu-Asp-Cys-Lys, (SEQ ID NO;14) the tetrapeptideN-Acetyl-Ser-Asp-Lys-Pro, and the tripeptide glutathione (Gly-Cys-γGlu).

The invention also includes a pharmaceutical composition comprising (a)INPROL and (b) at least one stimulatory compound selected from the groupconsisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11,IL-13, IL-14, IL-15, G-CSF, GM-CSF, M-CSF, erythropoietin,thrombopoietin, stem cell factor, and flk2/flt3 ligand.

The current invention describes an inhibitor of stem cells (INPROL)which is different from those known in the art such as MIP-1α, TGFβ, thetetrapeptide of Frindel and colleagues or the pentapeptide of Paukovitsand coworkers (cf., Wright & Pragnell, 1992 (op cit)). Naturallyoccuring INPROL has a molecular weight exceeding 10,000 daltons byultrafiltration which distinguishes it from the tetrapeptide as well asthe pentapeptide. It is more hydrophobic than MIP-1α or TGFβ, in reversephase chromatography systems, distinguishing it from those cytokines.Further, its mode of action is different from that of any previouslydescribed inhibitor in that it is active in an in vitro assay when usedduring a preincubation period only. MIP-1α for example, is not effectivewhen used during a preincubation period only (Example 5). Further,naturally occuring INPROL is active in an assay measuring "highproliferative potential cells" (HPP-PFC) whereas MIP-1α is not (Example6).

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-4 show an SDS polyacrylamide gel run of the product after eachstage of purification.

FIG. 1--Lane 1 is chymotrypsinogen, Lane 2 is ovalbumin, Lane 3 is BSA,Lane 4 is fractions <30 kD, Lane 5 is fractions 30-50 kD and Lane 6 isfractions 50-100 kD.

FIG. 2--Lane 1 is after ammonium sulfate precipitation (40-80%) andlanes 2-5 are DEAE fractions (Lane #2 represents the active fraction).

FIG. 3--Lane 1 is the supernatant after ammonium sulfate precipitation,Lane 2 is the active DEAE fraction, Lanes 3-5 represent gel filtrationfractions (lane #5 represents the active fraction)

FIG. 4--Lane 2 represents the final product.

FIG. 5 shows a reverse phase HPLC chromatogram of the finalpurification.

FIG. 6 shows tritiated thymidine incorporation (cpm) into cells of theFDCP-mix line without (Control=0% Inhibition) and with variousconcentrations of INPROL purified from porcine bone marrow (pINPROL).Data are normalized against the control value.

FIG. 7 shows the percent of cells in the S phase of the cell cycle aftertreatment of mice with testosterone propionate (TSP), TSP plus pINPROL,or vehicle (Control). Each group contained 25 animals (3-4 per timepoint).

FIG. 8 shows survival of mice treated with two doses of 5-FU, with orwithout pINPROL treatment. Each group contained 30 animals.

FIG. 9 shows survival of irradiated mice, with and without pINPROLtreatment. Each group contained 50 animals.

FIGS. 10A and 10B show regeneration of normal bone marrow long termculture cells 1 week (10A) and 3 weeks (10B) after treatment with Ara-Cor Ara-C plus pINPROL.

FIG. 11 shows survival of mice (75 per group) after lethal irradiationand transplantation of 3×10⁴ bone marrow cells after pre-incubation withmedium (Control) or pINPROL (25 ng/ml) for 4 hours. Survival wasmonitored for 30 days.

FIG. 12 shows the CFU-GM number formed after 14 days in culture by bonemarrow cells from mice after lethal irradiation and restoration withdonor bone marrow cells preincubated with pINPROL or medium for 4 hours.

FIG. 13 shows suspension cells from lymphoid long-term culture whichwere taken every week, washed out, and plated with IL-7 (10 ng/ml) afterpreincubation with medium or pINPROL for 4 hours.

FIG. 14 shows improved repopulating ability of leukemic peripheral bloodcells treated with pINPROL. Long term culture initiating cells (LTC-IC)were measured by plating adherent and nonadherent LTC cells with andwithout pINPROL, and scoring CFU-GM on day 7. Data are normalized tocontrol values.

FIG. 15A shows a C4 reverse phase chromatogram of purified pINPROLeluting at 53% acetonitrile. Lane 1 is the crude material, Lane 2 ismolecular weight markers and Lane 3 is the purified material. FIG. 15Bshows a C4 reverse phase chromatogram of MIP-1α eluting at 43.9%acetonitrile. FIG. 15C shows an SDS-PAGE chromatogram of the crudepINPROL preparation and of the purified preparation after reverse phase.

FIG. 16 shows hemoglobin sequences: FIG. 16A shows the cDNA (SEQ IDNO:15) and amino acid (SEQ ID NO:16) sequences of human alpha hemoglobinand FIG. 16B shows the cDNA (SEQ ID NO:17) and amino acid (SEQ ID NO:18)sequences of human beta hemoglobin. Numbering is according to the aminoacid. FIG. 16C shows an amino acid sequence comparison of the alpha andbeta chains of human, (SEQ ID NO:16 and SEQ ID NO:18) murine (SEQ IDNO:19 and SEQ ID NO:20) and porcine (SEQ ID NO:21 and SEQ ID NO:22)hemoglobins.

FIG. 17 shows a comparison of the C₄ reverse-phase HPLC traces ofpINPROL (FIG. 17A) and of crystallized pig hemoglobin (FIG. 17B).

FIG. 18 shows an SDS-PAGE gel of fractions from a C₄ reverse phase HPLCseparation of crystallized pig hemoglobin. Lane 1 shows molecular weightmarkers, Lane 2 shows Fractions 48-49, derived from the first peak (at47.11 min), Lane 3 shows fractions 50-51, derived from the second peak(at 49.153 min), Lane 4 shows fractions 54-55, derived from the thirdpeak (at 52.25 min) and Lane 5 shows fractions 56-57, derived from thefourth peak (at 53.613 minutes).

FIGS. 19A-B show a comparison of the 2-dimensional gel electrophoresesof pINPROL (FIG. 19A) and of purified pig beta hemoglobin (FIG. 19B).

FIG. 20 shows a comparison of the effects of purified pig alphahemoglobin, beta hemoglobin or pINPROL in the FDCP-MIX assay.

FIG. 21 shows the reverse phase separation of porcine hemoglobin using ashallow elution gradient.

In order that the invention herein described may be more fullyunderstood, the following detailed description is set forth. Thisdescription, while exemplary of the present invention, is not to beconstrued as specifically limiting the invention and such variationswhich would be within the purview of one skilled in this art are to beconsidered to fall within the scope of this invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

INPROL reversibly inhibits division of stem cells. Specifically, INPROLis effective in temporarily inhibiting cell division of hematopoieticstem cells. Thus, the method of this invention may be employed inalleviating the undesirable side effects of chemotherapy on thepatient's hematopoietic, myeloid and immune systems by protecting stemcells from damage caused by chemotherapeutic agents or radiation used todestroy cancer or virally infected cells. In one embodiment of theinvention, INPROL is administered to the patient in a dosage sufficientto inhibit stem cell division while the chemotherapeutic agent acts ondiseased cells. After the chemotherapeutic agent has performed itsfunction, the stem cells inhibited by INPROL will, without furthertreatment, revert to dividing cells. If it is desired to enhance theregeneration of hematopoiesis, stimulatory growth factors or cytokinesmay be used in addition.

As used herein, the term "INPROL" includes mammalian proteins, purifiedas in the Examples, hemoglobin, the alpha chain of hemoglobin (with orwithout the heme group), the beta chain of hemoglobin (with or withoutthe heme group), mixtures of alpha and beta chains (with or without theheme group), and fragments or analogs of these proteins includingembryonic, fetal or adult forms (e.g., alpha, beta, gamma, delta,epsilon or zeta chains, either alone or as mixtures, dimers ormultimers, with or without the heme group) having the ability to inhibitstem cell proliferation. The term "INPROL" includes naturally occurringas well as non-naturally occurring (e.g., recombinantly produced) formsof these proteins.

In a typical clinical situation, INPROL is administered to a patient ina daily regimen by intravenous injection or infusion in dosage unit formusing, for example, 0.01 to 100 mg/kg, advantageously 0.1 to 1.0 mg/kg,of INPROL administered, e.g., 4 to 60 hours prior to standardchemotherapy or radiation treatments.

In another embodiment of the invention, pretreatment with INPROL allowsfor increased doses of chemotherapeutic agents or of radiation beyonddoses normally tolerated in patients.

A large fraction of hematopoietic stem cells are normally quiescent(non-cycling). However, as a compensatory response tochemotherapy-induced hematopoietic damage, a larger proportion of stemcells enter into cycling after chemotherapy, which makes themparticularly vulnerable to subsequent doses of cytotoxic chemotherapy ortherapeutic irradiation. By inhibiting cycling of such stem cells,INPROL treatment permits earlier or more frequent administration ofsubsequent doses of cytotoxic chemotherapy, either at conventional orelevated doses.

In one embodiment of the invention, INPROL (0.1 mgs. to 6 gms -advantageously 1.0 to 60 mgs.) is administered about 24 hours to 10 daysafter an initial dose of chemotherapy. After another 4 to 60 hours,advantageously 24 to 48 hours, another dose of chemotherapy isadministered. This cycle of alternating chemotherapy and INPROL iscontinued according to therapeutic benefit. Chemotherapy agents andprotocols for administration are selected according to suitability forparticular tumor types in standard clinical practice. Optionally,stimulatory growth factors such as G-CSF, stem cell factor, are usedafter chemotherapy or radiation treatment to further improvehematopoietic reconstitution.

For ex vivo applications 0.1 ng to 100 ng/10⁶ cells/ml, advantageously20-50 ng/10⁶ cells/ml, of INPROL are used.

In another embodiment of the invention, INPROL is employed in a methodfor preparing autologous hematopoietic cells for transplantation. Thehematopoietic cells are treated ex vivo with an effective amount ofINPROL to inhibit stem cell division and then purged of cancerous cellsby administering to the marrow cultures an effective amount of achemotherapeutic agent or radiation. Chemotherapy agents withspecificity for cycling cells are preferred. Marrow thus treated isreinjected into the autologous donor. Optionally, the patient is treatedwith an agent known to stimulate hematopoiesis to improve thehematopoietic reconstitution of the patient.

In another embodiment of the invention, INPROL is employed as anadjunctive therapy in the treatment of leukemia. For example, in diseasestates where the leukemic cells do not respond to INPROL, the leukemichematopoietic cells cells are treated ex vivo with INPROL. Theproliferation of normal stem cells is prevented by administration ofINPROL. Thus, during the time that the proliferating leukemic cells aretreated with a cell cycle-specific cytotoxic agent, a population ofnormal stem cells is protected from damage. Additionally, a stimulatorycytokine, such as IL-3 or GM-CSF, is optionally administered to inducecycling in the leukemic cells during drug or radiation treatment whilethe normal stem cells are protected with INPROL. The patient is treatedwith chemotherapy agents or radiation to destroy leukemic cells, and thepurged marrow is then transplanted back into the patient to establishhematopoietic reconstitution.

Similarly, in another embodiment of the invention for treatment ofpatients with serious viral infections that involve blood cells orlymphocytes, such as HIV infection, hematopoietic cells are treated exvivo with INPROL followed by antiviral agents, drugs which destroyinfected cells, or antibody-based systems for removing infected cells.Following myeloablative antiviral or myeloablative chemotherapy toeradicate viral host cells from the patient, the INPROL-treated marrowcells are returned to the patient.

In another embodiment of the invention, INPROL is employed to treatdisorders related to hyperproliferative stem cells. For example,psoriasis is a disorder caused by hyperproliferating epithelial cells ofthe skin and is sometimes treated with cytotoxic drugs. Otherpre-neoplastic lesions in which stem cell proliferation is involved arealso amenable to effective amounts of INPROL employed to inhibit whollyor partially the proliferation of the stem cells. For these uses,topical or transdermal delivery compositions (e.g., ointments, lotions,gels or patches) containing INPROL are employed where appropriate, as analternative to parenteral administration. In most cases of leukemia, theleukemia progenitors are differentiated cell populations which are notaffected by INPROL and which are therefore treated by methods usingINPROL such as those described above. In cases where leukemiaprogenitors are very primitive and are directly sensitive to inhibitionby INPROL, proliferation of leukemia cells is attenuated byadministration of effective amounts of INPROL.

Antibodies, monoclonal or polyclonal, are developed by standardtechniques to the INPROL polypeptides. These antibodies or INPROLpolypeptides are labeled with detectable labels of which many types areknown in the art. The labeled INPROL or anti-INPROL antibodies are thenemployed as stem cell markers to identify and isolate stem cells byadministering them to a patient directly for diagnostic purposes.Alternatively, these labeled polypeptides or antibodies are employed exvivo to identify stem cells in a hematopoietic cell preparation toenable their removal prior to purging neoplastic cells in the marrow. Ina similar manner, such labeled polypeptides or antibodies are employedto isolate and identify epithelial or other stem cells. In addition,such antibodies, labeled or unlabeled, are used therapeutically throughneutralization of INPROL activity or diagnostically through detection ofcirculating INPROL levels.

INPROL can be cloned from human gene or cDNA libraries for expression ofrecombinant human INPROL using standard techniques. For example, usingsequence information obtained from the purified protein, oligonucleotideprobes are constructed which can be labeled, e.g., with 32-phosphorus,and used to screen an appropriate cDNA library (e.g., from bone marrow).Alternatively, an expression library from an appropriate source (e.g.,bone marrow) is screened for cDNA's coding for INPROL using antibody orusing an appropriate functional assay (e.g., that described in Example2). Hemoglobin itself, as well as the individual alpha and beta chains,have been cloned and expressed using methods known in the state of theart (see Pagnier et al., Rev. Fr. Transfus. Hemobiol. 35:407-15, 1992;Looker et al., Nature 356:258-60, 1992; Methods in Enzymology vol. 231,1994). Each of these articles is hereby incorporated by reference.

The present invention includes DNA sequences which include: theincorporation of codons "preferred" for expression by selectednonmammalian hosts: the provision of sites for cleavage by restrictionendonuclease enzymes; and the provision of additional initial, terminalor intermediate DNA sequences which facilitate construction ofreadily-expressed vectors or production or purification of the alpha,beta, gamma, delta, epsilon and/or zeta chain of hemoglobin.

The present invention also provides DNA sequences coding for polypeptideanalogs or derivatives of hemoglobin alpha, beta, gamma, delta, epsilonand/or zeta chains which differ from naturally-occurring forms in termsof the identity or location of one or more amino acid residues (i.e.,deletion analogs containing less than all of the residues specified;substitution analogs, wherein one or more residues specified arereplaced by other residues; and addition analogs wherein one or moreamino acid residues is added to a terminal or medial portion of thepolypeptide) and which share some or all of the properties ofnaturally-occurring forms.

In an advantageous embodiment, INPROL is the product of prokaryotic oreukaryotic host expression (e.g., by bacterial, yeast, higher plant,insect and mammalian cells in culture) of exogenous DNA sequencesobtained by genomic or cDNA cloning or by gene synthesis. That is, in anadvantageous embodiment, INPROL is "recombinant INPROL". The product ofexpression in typical yeast (e.g., Saccharomyces cerevisiae) orprokaryote (e.g., E. coli) host cells are free of association with anymammalian proteins. The products of expression in vertebrate(e.g.,non-human mammalian (e.g., COS or CHO) and avian) cells are freeof association with any human proteins. Depending upon the hostemployed, polypeptides of the invention may be glycosylated or may benon-glycosylated. Polypeptides of the invention optionally also includean initial methionine amino acid residue (at position -1).

The present invention also embraces other products such as polypeptideanalogs of the alpha, beta, gamma, delta, epsilon and/or zeta chain ofhemoglobin. Such analogs include fragments of the alpha, beta, gamma,delta, epsilon and/or zeta chain of hemoglobin. Following well knownprocedures, one can readily design and manufacture genes coding formicrobial expression of polypeptides having primary sequences whichdiffer from that herein specified for in terms of the identity orlocation of one or more residues (e.g., substitutions, terminal andintermediate additions and deletions). Alternatively, modifications ofcDNA and genomic genes can be readily accomplished by well-knownsite-directed mutagenesis techniques and employed to generate analogsand derivatives of the alpha, beta, gamma, delta, epsilon or zeta chainsof hemoglobin. Such products share at least one of the biologicalproperties of INPROL but may differ in others. As examples, products ofthe invention include the alpha, beta, gamma, delta, epsilon or zetachains which is foreshortened by e.g., deletions; or those which aremore stable to hydrolysis (and, therefore, may have more pronounced orlonger-lasting effects than naturally-occurring); or which have beenaltered to delete or to add one or more potential sites forO-glycosylation and/or N-glycosylation or which have one or morecysteine residues deleted or replaced by, e.g., alanine or serineresidues and are more easily isolated in active form from microbialsystems; or which have one or more tyrosine residues replaced byphenylalanine and bind more or less readily to target proteins or toreceptors on target cells. Also comprehended are polypeptide fragmentsduplicating only a part of the continuous amino acid sequence orsecondary conformations within the alpha, beta, gamma, delta, epsilon orzeta chains which fragments may possess one property of INPROL (e.g.,receptor binding) and not others (e.g., stem cell inhibitory activity).It is noteworthy that activity is not necessary for any one or more ofthe products of the invention to have therapeutic utility (see, Weilandet al., Blut 44:173-5, 1982) or utility in other contexts, such as inassays of inhibitory factor antagonism. Competitive antagonists areuseful in cases of overproduction of stem cell inhibitors or itsreceptor.

In addition, peptides derived from the protein sequence which retainbiological activity can be chemically synthesized using standardmethods. The present invention also provides for sequences coding forpeptide analogs or derivatives of hemoglobin alpha, beta, gamma, delta,epsilon and/or zeta chain which differ from naturally-occurring forms interms of the identity or location of one or more amino acid residues(e.g., deletion analogs containing less than all of the residuesspecified; substitution analogs, wherein one or more residues specifiedare replaced by other residues, either naturally occuring or otheranalogs known in the state of the art such as D-amino acids; andaddition analogs wherein one or more amino acid residues is chemicallymodified to increase stability, solubility and/or resistance toproteolysis) and which share some or all of the properties ofnaturally-occurring forms.

Peptide sequences such as described above can be identified by a varietyof means. Comparison of the three dimensional structures of nativehemoglobin chains active in the assay (e.g., alpha chain) withstructurally related proteins which are inactive (e.g., myoglobin) canidentify regions which have different conformations in three-dimensionalspace and which are therefore candidate regions for active peptides.Another approach uses selective proteolysis, in which proteolyticenzymes are used in limited digests of hemoglobin chains resulting inpeptides which can separated, for example, by reverse phase HPLC andthen assayed for stem cell inhibition. Peptides can also be generated bychemical synthesis (e.g., solid phase synthesis); a series ofoverlapping peptides (e.g., 15-mers) which encompass the sequence of thehemoglobin chain of interest (e.g., alpha chain) can easily be generatedand tested in stem cell assays. Combinatorial libraries can be generatedin which multiple chemical syntheses are conducted and where selectedamino acid positions are made variable resulting in large numbers ofpeptide analogs for screening (e.g., Dooley et al., Peptide Research8:124-137, 1995). Alternatively, recombinant methods can be employed.Site directed mutagenesis can be used to identify critical residuesnecessary for activity of a particular hemoglobin chain. Regions of achain which is known to be active as a stem cell inhibitor (e.g., alphachain) can be substituted with regions from a related but inactiveprotein (e.g., myoglobin) and tested in stem cell assays, allowing foridentification of regions necessary for activity. Such identifiedregions can be expressed as peptides and tested for activity in stemcell cycling assays.

Homologous or analogous versions of INPROL from other species areemployed in various veterinary uses, similar to the therapeuticembodiments of the invention described above.

INPROL acts on cycling stem cells by reversibly placing them in anundividing "resting" state. When it is desirable to stimulate thequiescent stem cells into division, e.g., after treatment of a patientwith cancer chemotherapy agents or radiation, colony-stimulating factorsand other hematopoietic stimulants are administered to the subject.Examples of such factors include but are not limited to: M-CSF (CSF-1),GM-CSF, G-CSF, Megakaryocyte-CSF, thrombopoieitin, stem cell factor orother cytokines, such as IL- 1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-9, IL-11, IL-12, IL- 13, IL- 14, or erythropoietin .

INPROL polypeptides or active fragments having stem cell inhibitoryactivity are purified or synthesized by conventional chemical processescombined with appropriate bioassays for stem cell inhibitory activity,as exemplified in the protocols described below.

In one embodiment of the invention, a therapeutically effective amountof the INPROL protein or a therapeutically effective fragment thereof isemployed in admixture with a pharmaceutically acceptable carrier. ThisINPROL composition is generally administered by parenteral injection orinfusion. Subcutaneous, intravenous, or intramuscular injection routesare selected according to therapeutic effect achieved.

When systemically administered, the therapeutic composition for use inthis invention is in the form of a pyrogen-free, parenterally acceptableaqueous solution. Pharmaceutically acceptable sterile protein solution,having due regard to pH, isotonicity, stability, carrier proteins andthe like, is within the skill of the art.

Also comprehended by the invention are pharmaceutical compositionscomprising therapeutically effective amounts of polypeptide products ofthe invention together with suitable diluents, preservatives,solubilizers, emulsifiers, adjuvants and/or carriers useful in INPROLtherapy. A "therapeutically effective amount" as used herein refers tothat amount which provides a therapeutic effect for a given conditionand administration regimen. Such compositions are liquids, gels,ointments, or lyophilized or otherwise dried formulations and includediluents of various buffer content (e.g., Tris-HCl, acetate, phosphate),pH and ionic strength, additives such as albumin or gelatin to preventadsorption to surfaces, detergents (e.g., Tween 20, Tween 80, PluronicF68, bile acid salts), solubilizing agents (e.g., glycerol, polyethyleneglycol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite),preservatives (e.g., Thimerosal, benzyl alcohol, parabens), bulkingsubstances or tonicity modifiers (e.g., lactose, mannitol), covalentattachment of polymers such as polyethylene glycol to the protein,complexation with metal ions, or incorporation of the material into oronto particulate preparations of polymeric compounds such as polylacticacid, polyglycolic acid, hydrogels, etc. or into liposomes, niosomes,microemulsions, micelles, unilamellar or multilamellar vesicles,biodegradable injectable microcapsules or microspheres, or proteinmatrices, erythrocyte ghosts, spheroplasts, skin patches, or other knownmethods of releasing or packaging pharmaceuticals. Such compositionswill influence the physical state, solubility, stability, rate of invivo release, and rate of in vivo clearance of INPROL. Controlled orsustained release compositions include formulation in lipophilic depots(e.g., fatty acids, waxes, oils). Also comprehended by the invention areparticulate compositions coated with polymers (e.g., poloxamers orpoloxamines) and INPROL coupled to antibodies directed againsttissue-specific receptors, ligands or antigens or coupled to ligands oftissue-specific receptors. Other embodiments of the compositions of theinvention incorporate particulate forms of protective coatings, proteaseinhibitory factors or permeation enhancers for various routes ofadministration, including parenteral, pulmonary, nasal, topical (skin ormucosal) and oral. In another embodiment, the composition containingINPROL is administered topically or through a transdermal patch.

In one embodiment, the compositions of the subject invention arepackaged in sterile vials or ampoules in dosage unit form.

The invention also comprises compositions including one or moreadditional factors such as chemotherapeutic agents (e.g., 5-fluorouracil(5FU), cytosine arabinoside, cyclophosphamide, cisplatin, carboplatin,doxyrubicin, etoposide, taxol, alkylating agents), antiviral agents(e.g., AZT, acyclovir), TNF, cytokines (e.g., interleukins),antiproliferative drugs, antimetabolites, and drugs which interfere withDNA metabolism.

The dosage regimen involved in a method for treating the subjectanticipating exposure to such cytotoxic agents or for treatment ofhyperproliferating stem cells is determined by the attending physicianconsidering various factors which modify the action of drugs; e.g., thecondition, body weight, sex, and diet of the patient, the severity ofany infection, time of administration and other clinical factors.

Following the subject's exposure to the cytotoxic agent or radiation,the therapeutic method of the present invention optionally employsadministering to the subject one or more lymphokines, colony stimulatingfactors or other cytokines, hematopoietins, interleukins, or growthfactors to generally stimulate the growth and division of the stem cells(and their descendants) inhibited by the prior treatment with INPROL.Such therapeutic agents which encourage hematopoiesis include IL1, IL2,IL-3, IL-4, IL-5, IL-6, IL-7, Meg-CSF, M-CSF (CSF-1), GM-CSF, G-CSF orerythropoietin. The dosages of these agents are selected according toknowledge obtained in their use in clinical trials for efficacy inpromoting hematopoietic reconstitution after chemotherapy orhematopoietic stem cell transplant. These dosages would be adjusted tocompensate for variations in the physical condition of the patient, andthe amount and type of chemotherapeutic agent or radiation to which thesubject was exposed. Progress of the reversal of the inhibition of thestem cells caused by administration of INPROL in the treated patient ismonitored by conventional methods.

In the treatment of leukemia, it is beneficial to administer both INPROLto inhibit normal stem cell cycling and a stimulator of leukemic cellgrowth, such as IL-3 or GM-CSF, simultaneously with the cytotoxic drugtreatment or during irradiation. By this protocol, it is possible toachieve the greatest differences between the cycling statuses and drugsensitivities of normal and leukemic cells.

EXAMPLE 1 In Vivo Stem Cell Proliferation Inhibition Assay

For the detection of stem cells proliferation the number of CFU-S inS-phase of the cell cycle was measured by the ³ H-Thymidine "suicide"method (Becker et al., Blood 26:296-308, 1965).

Immature hematopoietic progenitors--Colony Forming Units in spleen(CFU-S)--can be detected in vivo by forming macroscopic colonies in thespleens of lethally irradiated mice, 8-12 days after the intravenousinjection of hematopoietic cells (Till & McCulloch, 1961).

For the standard CFU-S proliferation assay the method of ³ H-Thymidine"suicide" is usually applied (Becker et al., 1965). The method is basedon incorporation of radiolabelled Thymidine, (³ H-Thymidine) a precursorof DNA into cells during DNA synthesis. The CFU-S which are in S-phaseof the cycle at the time of testing, are killed by the highradioactivity and therefore not able to form colonies in spleen. Thus,the difference between the number of CFU-S formed by the injection ofthe cell sample incubated without ³ H-Thymidine and the same cellsincubated with ³ H-Thymidine shows the percentage of the proliferatingCFU-S in the original sample.

The inhibitor testing can not be done with the bone marrow stem cellpopulation from unstimulated animals, as far as the inhibitor onlyeffects cycling CFU-S, which are as low as 7-10% of the total CFU-Spopulation in the bone marrow of normal mice.

To stimulate CFU-S proliferation, phenylhydrazine (PHZ), or sublethalirradiation were used (Lord, 1976).

We have developed the method of using testosterone-propionate (TSP)based on its stimulatory effect on CFU-S cycling (Byron et al., Nature228:1204, 1970) which simplified the testing and did not cause any sideeffects. The TSP induced stimulation of CFU-S proliferation within 20-24hours after injection and the effect could be seen for at least 7 days.

The procedure used for the screening of the fractions duringpurification of the Inhibitor was as follows:

Mice: BDF₁ or CBF₁ , mice strains were used throughout all testing.

Donor mice were treated with a 10 mg/100 g dose of TSP byintraperitoneal injection of 0.2 ml/mouse in order to induce 30-35% ofthe CFU-S into S-phase.

Twenty-four hours later the bone marrow is taken from the femurs for thecell suspension preparation. Five to ten million cells per ml are thenincubated with different control and test fractions for 3.5 hours at 37°C. in water bath, with two tubes for each group (one for hot(radioactive) and one for cold (non-radioactive)).

After 3.5 hours, ³ H-Thymidine (1 mCi/ml, specific activity 18-25Ci/mmole) is added to each hot tube in a volume of 200 μl per 1 ml ofcell suspension; nothing is added to the cold tubes. Incubation iscontinued for 30 more minutes at 37° C.

After the 30 minute incubation, the kill reaction is terminated byadding 10 ml of cold (4° C.) medium containing 400 μg/ml nonradioactiveThymidine. Cells are washed extensively (3 times).

Cells are resuspended and diluted to a desirable concentration for theinjections, usually 2-4×10⁴ cells per mouse in 0.3-0.5 ml.

Recipient mice, 8-10 per group, are irradiated not later than 6 hoursbefore the injections.

Recipient spleens are harvested on day 9-12 and fixed in Tellesnitsky'ssolution; the colonies are scored by eye score. The percentage of cellsin S-phase are calculated using the formula. ##EQU1## where a--CFU-Snumber without ³ H-Thymidine where b--CFU-S number with ³ H-Thymidine

The test data of INPROL presented in Table 1 demonstrate that cyclingstem cells after treatment with INPROL become resistant to the action of³ H-Thymidine. For this and all of the following examples, the term"pINPROL" refers to the purified protein from porcine bone marrow. Thesame protection is seen for the S-phase specific cytotoxic drugscytosine arabinoside and hydroxyurea (data not shown). If the treatedstem cells are then washed with the cold media containingnon-radioactive Thymidine, the surviving stem cells proliferate in mousespleens to form colonies normally.

                  TABLE 1    ______________________________________    Inhibitory Activity Of pINPROL On CFU-S Proliferation    During Four Hour Incubation With Bone Marrow Cells                                    Percent CFU-S    Group      -.sup.3 H-TdR                          +.sup.3 H-TdR                                    Killed by .sup.3 H-TdR    ______________________________________    No incubation               22.2 ± 2.0*                          13.7 ± 2.4*                                    38.3 ± 1.7    4 Hour with Media               18.7 ± 3.0*                          11.4 ± 1.3*                                    43.1 ± 1.4    4 Hour with               21.2 ± 2.3*                          20.7 ± 2.6*                                     2.1 ± 0.08    pINPROL    ______________________________________     *CFU-S per 2 × 10.sup.4 cells

EXAMPLE 2 In Vitro Stem Cell Proliferation Inhibition Assay

Using the following test system (Lord et al., in The Inhibitors ofHematopoiesis pp. 227-239, 1987) the direct effect of INPROL was shown.The multilineage factor (IL-3) dependent stem cell line, FDCP mix A4(A4), was maintained in IMDM medium supplemented with 20% horse serumand 10% WEHI-3-conditioned medium as a source of colony-stimulatingIL-3.

A tritiated Thymidine incorporation assay was used to measureproliferation: A4 cells (5×10⁴ in 100 μl medium with 20% horse serum and50% of WEHI-3 CM) were incubated at 37° C. in 5% CO₂ for 16 hours.

pINPROL or the crude BME (fraction IV) were added at the start.Tritiated thymidine ((³ H-Tdr) 3.7KBq in 50 μl at 740 GBq/mmole) wasthen added to each group for a further 3 hours of incubation. The levelof proliferation was measured by harvesting cells and the % inhibitoncalculated using the formula ##EQU2##

Incorporation of tritiated thymidine (³ H-Tdr) by FDCPmix-A4 cells grownin the presence of graded doses of normal bone marrow extract or pINPROLis depicted on FIG. 6. It can be seen that purified composition ofpINPROL is at least 1,000 times more active than the starting material.Time of exposure (16 hours) is an important factor for effectiveinhibition and shows the evidence of the direct effect of pINPROL onstem cells of the A4 cell line.

EXAMPLE 3 Inhibition of CFU-S Proliferation by INPROL Injected in vivo:Doses and the Duration of the Effect

The studies of the effect of INPROL injected in vivo revealed thatINPROL can effectively block the recruitment of CFU-S into cycle, thusprotecting those cells from the cytotoxic effect of further treatment,showing its potential for clinical use.

The experimental protocol had two goals: to check the effect of INPROLon CFU-S when injected in vivo and to define the effective duration ofINPROL activity in relation to cycling stem cells.

To stimulate CFU-S proliferation, the injection oftestosterone-propionate was used based on the effect mentioned above inExample 1.

Mice BDF1 were injected with TSP (10 mg/100 g) on Day 0; 24 hours latermice of each experimental group (4 mice per group) received a singlepINPROL injection at doses of 0 μg, 5 μg, 10 μg, and 15 μg/mouse i.p.

Twenty-four hours after pINPROL injection, mice were sacrificed and thepercent of cycling CFU-S was measured by the assay described inExample 1. TSP injection induced about 50% CFU-S into cycling incomparison with 7% in untreated mice. pINPROL in doses as low as 2μg/mouse was able to inhibit TSP induced proliferation down to thenormal level.

For the duration of the effect evaluation, one group of mice (21 miceper group) was injected with TSP only and another group was injectedboth with TSP and pINPROL (24 hours after TSP). The CFU-S cycling wasmeasured every 24 hours during a week by taking 3 donors from each groupand measuring CFU-S cycle status in their bone marrow by methoddescribed (see Example 1). Data presented in FIG. 7 show that while theduration of the effect of TSP is at least 7 days, a single injection ofINPROL can place CFU-S into quiescence and keep them out of cycle for nomore than 48-72 hours. Since the majority of chemotherapeutic agentsused for cancer and leukemia chemotherapy have a relatively short invivo half-life, usually less than 24 hours, the INPROL effect accordingto the data obtained is maintained for longer than the effective timeduring which the chemotherapeutic agents like cytosine arabinoside orhydroxyurea are active in vivo. More importantly, for chemotherapeuticand radiation treatments having longer intervals (more than 24 hours andless than 96 hours) between the first (non-damaging for the stem cells)and the second (damaging to the CFU-S) treatments, a single injection ofINPROL during the intervals between the two applications ofchemotherapeutic agent or radiation should be sufficient. For severalrepeatable cycles of cytotoxic therapy or radiation the same strategycould be applied based on the duration of the INPROL effect.

EXAMPLE 4 Most Primitive Hematopoietic Stem Cells Stimulated to CycleRapidly After Treatment with 5-FU are Protected by INPROL from theSecond 5-FU Exposure

The drug 5-fluorouracil (5-FU) drastically reduces the number of cellsin the myeloid and lymphoid compartments. It is usually thought of asbeing cell-cycle specific, targeting rapidly proliferating cells,because incorporation of the nucleotide analogue into DNA during S-phaseof the cell cycle or before results in cell death. The long-termsurvival and immunohematopoietic reconstitution of the bone marrow ofmice is not affected by a single dose of 5-FU; however, it wasdemonstrated (Harrison et al. Blood 78:1237-1240, 1991) that pluripotenthematopoietic stem cells (PHSC) become vulnerable to a second dose of5-FU for a brief period about 3-5 days after the initial dose. It can beexplained that PHSC normally cycle too slowly for a single dose of 5-FUto be effective and are stimulated into rapid cycling by stimuliresulting from the initial 5-FU treatment. We have proposed that PHSCcan be returned to a slow cycle status by INPROL and thus protected fromthe second 5-FU treatment.

The mice used in these experiments were BDF1 male mice. A stock solutionof 5-FU (Sigma) was prepared in physiologic saline at a concentration of10 μg/ml. Each treated mouse received 2 mg of 5-FU per 10 g body weightvia a tail vein at Day 0 of the experiment; 24 hours later mice wereinjected with pINPROL (10 μg/100 g of body weight) intraperitoneally andon Day 3 were injected with the second dose of 5-FU. The survival studywas conducted by monitoring the death of mice in experimental (treatmentwith pINPROL) and control groups of 30 mice each. The survival curvesare shown in FIG. 8.

EXAMPLE 5 Effects of Pre-Incubation with INPROL vs. MIP-1α in BoneMarrow Cells

The purpose of this experiment was to compare the inhibitory effects ofpre-incubation with pINPROL and MIP-1α on mouse bone marrow cells invitro.

The following procedure was used:

in vivo: BDF1 mice, 6-15 weeks of age, are injected with 200 mg/kg 5FUi.p. 48 hours before harvesting marrow from the femurs.

in vitro: A single cell pooled suspension is counted and 5×10⁶ cells areincubated in a total of 2 mls with or without pINPROL or MIP-1α, with 5%horse serum, IMDM media with added L-glutamine, at 37° C. and 5% CO₂ for4 hours. The cells are then washed twice and recounted. They are platedin methylcellulose in the following final conditions:

0.8% methylcellulose

25% horse serum

20 ng/ml recombinant murine IL3

L-glutamine added

5×10⁵ cells per ml

IMDM media

Plates were incubated for 11 days at 37° C. and 5% CO₂ in 100% humidity.Colonies more than 50 cells were counted.

                  TABLE 2    ______________________________________    Groups     Colony Number                           Percent of Control    ______________________________________    Control    31.0        100%    pINPROL    21.25       68.5%    MIP-1α               35.25       114%    ______________________________________

EXAMPLE 6 INPROL inhibits HPP-CFC proliferation

An in vitro assay for assessing murine reconstituting stem cells andearly precursors is the high proliferative potential colony (HPP-PFC)assay; other related assays, e.g., CFU-A, CFU-GM, CFU-E, and CFU-GEMM,detect progressively restricted progenitor populations (M. Moore, Blood177:2122-2128, 1991). This example shows that pretreatment of cells withpINPROL inhibits their proliferation, whereas MIP-1α fails to do sounder these experimental conditions.

BDF1 mice were treated with 5-fluorouracil (200 mg/kg i.p.) before theirbone marrow was assayed for HPP-CFC numbers. Cells were washed bycentrifugation and incubated at densities of 10⁶ to 5×10⁶ /ml in mediumwith either no added agent (Controls), pINPROL (25 ng/ml) or MIP-1α (200ng/ml) for 4 hours. After incubation, cells were washed and plated inagar (0.3%) with 30% FCS and combined conditioned medium from 5637 andWEHI-3B cell lines (7.5% of each conditioned medium, as recommended bySharp et al., 1991). Plating concentration was 5×10⁴ cells/ml in 60 mmdishes. Colonies were scored on day 14 and the results are indicatedbelow.

                  TABLE 3    ______________________________________    Group         HPP-CFU  % of Control    ______________________________________    Control       15.5 ± 1.2                           100%    pINPROL        8.3 ± 0.7                           53.5%    MIP-1α  15.8 ± 0.9                           101%    ______________________________________

According to these results, MIP-1α did not inhibit proliferation of themost immature precursors when present only during the pre-incubationperiod. pINPROL did effectively inhibit proliferation under theseconditions, indicating fundamental differences between pINPROL andMIP-1α in terms of biological activity.

EXAMPLE 7 INPROL Therapy Effect on the Recovery from Radiation-inducedBone Marrow Aplasia

Bone marrow aplasia is the primary limiting toxicity of radiation cancertherapy. It has been demonstrated that some growth factors (e.g., G-CSF,GM-CSF, erythropoietin) can accelerate recovery from radiation-inducedbone marrow aplasia. The concept of protection by using an inhibitor ofstem cell proliferation is a different and complementary approach incoping with hematological damage. To follow the treatment proceduredeveloped earlier (Examples 3, 4) a model of lethal irradiation of micewas established. It is known in the art that mice receiving 9Gy ofcobalt 60 start dying after 10-14 days; by Day 30, mortalityapproximates 50%. This lethal dose was used in our model by splitting itinto two subsequent applications of 4.5Gy each with an interval 3 daysbetween doses. Preliminary data showed that the survival curve in thatmodel was very close to that known for a single irradiation with 9Gy;moreover the test for the CFU-S proliferation showed that 24 hours afterthe first irradiation, 35-50% of CFU-S are induced to proliferate. Suchcells can be protected by a stem cell inhibitor delivered prior to thesecond dose.

To examine this possibility, mice (50 mice/group) received 4.5Gy on Day0. Twenty four hours later, one group received pINPROL (2 μg/mouse i.p.)and another, control group was injected with saline. The second dose ofradiation (4.5 Gy) was given on Day 3.

FIG. 9 shows the increased survival after a single dose of pINPROL. Theconditions of the model are clinically relevant for treating any cancer,including those characterized by solid tumors; such treatment would beadministered to a patient having cancer by delivering an effective doseof INPROL between two consecutive dosages of radiation, thereby allowinggreater dosages of radiation to be employed for treatment of the cancer.It should also be possible to extend this modality to chemotherapeuticagents.

EXAMPLE 8 INPROL Use for the Autologous Bone Marrow Transplantation

Bone marrow transplantation is the only known curative therapy forseveral leukemias (CML, AML, and others). Ex vivo conditioning ofautologous BMT for infusion should provide potential autologous sourcesof normal stem cells free of leukemic contamination and able torepopulate the recipient's hematopoietic system to allow aggressive andeffective therapy.

1. Long-term Bone Marrow Culture L1210 Leukemia Model For The Study OfINPROL Effect Preserving Normal Hematopoiesis During Purging With AraC

Long-Term Bone Marrow Cultures (LTBMC) were established according toToksoz et al. (Blood 55:931-936, 1980) and the leukemic cell line L1210was adopted to the LTBMC by co-cultivation during 2 weeks. Thesimultaneous growth of normal and leukemic progenitors occurred in thesecombined LTBMC/L1210 cultures, similar to the situation in the bonemarrow of a leukemic patient. Discrimination between normal colonyforming units CFU and leukemic CFU was possible by growing them as agarcolonies in the presence or absence of the conditioned medium fromWEHI-3 (a murine IL-3 producing cell line). Normal cells undergoapoptosis in the absence of IL-3 whereas leukemic cells can formcolonies in its absence. Suspension cells from LTBMC-L1210 compositiongive approximately 150 colonies in presence of IL-3 (normalhematopoietic clones) and 70 colonies when growing without IL-3(leukemic clones) per 50,000 cells plated.

The procedure of purging was as follows: on Day 0 all suspension cellsand media (10 ml/flask) were taken off the flasks with LTBMC-L1210 andreplace with 2 ml of media containing 200 μg cytosine arabinoside (AraC)(Tsyrlova et al. in Leukemia: Advances in Biology and Therapy v. 35,1988); after 20 hours of incubation, flasks were washed out and replacedwith 2 ml of fresh media alone (control group) or media containingpINPROL at 25 ng/ml for 4 hours. After this preincubation, cells wereincubated again with 100 μg/flask AraC for 3 hours at 37° C. Each groupcontained 4 flasks. LTBMC-L1210 cultures were washed 3 times andreplaced with fresh LTBC media; they were maintained as before for theregeneration studies for 3-4 weeks.

Data are presented in FIG. 10. There was no cell growth seen in controlcultures treated with AraC only, while in INPROL protected flasksregeneration of hematopoiesis occurred much more rapidly due toproliferation of progenitors from the adherent layer. Moreover, thecells from the experimental group when plated in agar grew only in thepresence of IL-3 giving about 100 CFU per 50,000 cells; no leukemic cellgrowth was observed at least during 4 weeks. Thus, marrow treated exvivo with an effective dose of AraC in combination with INPROL can bepurged of cancerous cells while the stem cells are be protected. Itshould be possible to extend this modality to other forms ofchemotherapy or radiation treatments.

2. Marrow Repopulating Ability (MRA) And Thirty Days Radioprotection AreIncreased By INPROL Treatment In Vitro

MRA, the ability of cells to repopulate the bone marrow of lethallyirradiated mice, together with the ability to confer radioprotection for30 days, is a direct in vivo measurement of the potential to rescuemyelosuppressed animals (Visser et al. Blood Cells 14:369-384, 1988).

For radioprotection studies BDF1 mice were irradiated with 9.5Gy andrestored by transplantation of bone marrow from testosterone-stimulateddonors. One group of recipients was restored by bone marrow cellspreincubated for 4 hours with medium (controls--group A) and another(group B) with 25 ng/ml pINPROL. Cells in both groups were washed and30,000 cells per mouse were transplanted into irradiated animals. Thesurvival data are shown (FIG. 11 ). The sum of 3 experiments isdepicted, with controls normalized to 100%. pINPROL incubation increasedthe survival of mice from 36.5% in control group up to 61.8% by Day 30.

The increase of MRA induced by preincubation with INPROL could be one ofthe mechanisms in the improving of the radioprotection. To examine thishypothesis, MRA was measured according to Visser et al. (op. cit.).Briefly, the donor BDF1 mice were pretreated with testosterone, theirbone marrow was preincubated with medium or pINPROL for 4 hours andinjected into irradiated animals. On Day 13, the bone marrow cells fromrecipient femurs were plated in agar in 3 different concentration (0.01,0.05, 0.1 equivalent of a femur) in the presence of 20% of horse serumand 10% of WEHI-CM. The number of Day 7 colonies represented the MRA asfar as the colony-forming cells in the bone marrow of recipients at thetime were the progenitors of the donor's immature stem cells.

As can be seen on FIG. 12 the MRA of the preincubated with INPROL cellpopulation is greater than in the control group (B).

EXAMPLE 9 Antihyperproliferative Effect Of INPROL On Stem Cells CanChange Their Differentiation Abnormalities

Hyperproliferation of CFU-S is not only seen during restoration fromcytotoxic drugs or irradiation but also as a consequence of normalaging, and is thought to be a major feature in Myelodysplastic Syndrome(MDS). It is accompanied by the differentiation disturbances such as aprevalence of the erythroid differentiation while the differentiationalong the granulocytic pathway is reduced.

Bone marrow cells were incubated for 4 hours at 37° C. with 25 ng/ml ofpINPROL or media (Control), washed and then plated in agar with 20% ofhorse serum, 2U/ml Erythropoietin, and 10% WEHI-CM. The number of BFU-Eand GM-CFU colonies were scored on Day 7. Data presented in Table 4 aresummarized from 3 experiments--4 animals per point were taken for eachgroup; 4 dishes were plated.

As is obvious from Table 4, the incubation of normal bone marrow (NBM)from intact young animals (BDF1 8-12 weeks old) with INPROL did notchange the number or proportion of different types of colonies. BDF₁donors pretreated with Testosterone Propionate (TSP) showed the sameincrease in CFU-S proliferation as was seen before (Example 1, 3, 4) aslight increase in the erythroid progenitor number (BFU-E colonies) anda decrease in GM-CFU, which were completely abrogated by the incubationwith INPROL. In addition, the abnormally high level of CFU-Sproliferation was returned to 10% of CFU-S in S-phase of cell cycle.CFU-S hyperproliferation is known to be a feature of some mouse strainssusceptible to viral leukemia induction, for example Balb/c mice (Table4), and can also be observed in older animals (Table 4). The sameredistribution of committed progenitors seen in TSP treated BDF1 mice isobserved in Balb/c and in older (23-25 month old) BDF1, which have incommon the abnormally high level of CFU-S proliferation. The correctionof both the proliferation of CFU-S and the differentiation was inducedby the incubation with INPROL. What is even more clinically relevant,the study showed that the in vivo injection of INPROL (2 μg/mouse)affected both proliferation of CFU-S and the ratio of erythroid (BFU-E)and GM-colonies (Table 4).

                  TABLE 4    ______________________________________    INPROL Effect On CFU-S Differentiation    Into Committed Progenitors BFU-E and CFU-GM                      Percent    Donors Of         CFU-S    Bone              Killed by    Marrow   pINPROL  .sup.3 HTdR                                BFU-E   CFU-GM    ______________________________________    BDF.sub.1 Young             -        12.0 ± 0.3                                28.33 ± 1.91                                        46.22 ± 3.44             +        15.0 ± 1.3                                22.00 ± 3.74                                        47.70 ± 3.72    BDF.sub.1 Old             -        47.1 ± 1.9                                43.75 ± 1.54                                         24.0 ± 1.33             +        11.4 ± 0.7                                15.25 ± 1.45                                         44.0 ± 7.63    BDF.sub.1             -        53.2 ± 1.6                                32.67 ± 2.44                                        15.71 ± 2.28    Stimulated             +         7.2 ± 0.4                                12.00 ± 1.83                                        35.50 ± 1.4    by TSP    Balb/C   -        57.0 ± 1.9                                47.60 ± 2.96                                        33.57 ± 3.45             +        23.0 ± 2.4                                24.86 ± 2.53                                        70.60 ± 4.96    ______________________________________

EXAMPLE 10 Immunostimulatory Activity of INPROL

It has been observed that the incubation of bone marrow cells containinga high proportion of proliferating CFU-S with INPROL not only changesthe cycling of CFU-S, but also their differentiation, switching thepredominantly erythroid differentiation in favor of granulocytic andlymphoid progenitors. This property of INPROL is of importance due tothe immunosuppression side effects of cytotoxic chemotherapy orradiotherapy, as well as the immunosuppression accompanyinghyperproliferative stem cell disorders and aging.

The example shows the direct effect of INPROL on the differentiation ofimmature precursors from the Lymphoid Long Term Culture (LLTC)established according to Wittlock & Witte (Ann. Rev. Immun. 3:213-35,1985) into pre-B progenitors, measured by the formation of colonies inmethylcellulose containing IL-7.

LLTC were established as described and fed with fresh LLTC-media (TerryFox Labs., Vancouver, Canada) twice a week. Nonadherent cells wereharvested once a week, washed free of factors and incubated for 4 hourswith 25 ng/ml pINPROL or medium alone for control. After the incubation,the cells were washed and plated at a concentration of 10⁵ cells/ml inmethylcellulose, containing 30% FCS, and 10 ng/ml of IL-7. Data from 3weeks are shown in FIG. 13. The number of large pre-B colonies varied incontrol, increasing with time, but preincubation with INPROL alwaysstimulated the growth of colonies 4 to 8 fold above the control level.This demonstrates an immunostimulatory property of INPROL which is ofuse in correcting immunodeficient states and in increasing desiredimmune responses, e.g., to vaccination.

EXAMPLE 11 INPROL Improves Repopulating Ability of Stem Cells--Long TermCulture Initiating Cells from Patient with CML

Chronic myeloid leukemia (CML) is a lethal malignant disorder of thehematopoietic stem cell. Treatment of CML in the chronic phase withsingle-agent chemotherapy, combination chemotherapy, splenectomy, orsplenic irradiation may control clinical signs and symptoms, but doesnot significantly prolong survival. As CML progresses from the chronicto the accelerated stage, standard therapy is not effective. At present,bone marrow transplantation (BMT) is the only known curative therapy forCML. Therapy with unrelated donor BMT is difficult due tohistoincompatibility problems. Fewer than 40% of otherwise eligible CMLpatients will have a suitably matched related donor; thereforeautologous transplantation is preferred. Ex vivo conditioning ofautologous BMT for infusion together with the ability to selectnon-leukemic (Ph-negative) myeloid progenitors from Ph-positive patientsgrowing in Long Term Culture (LTC) suggest the potential of autologoussources of normal stem cells to allow aggressive and effective therapyof CML.

In the context of BMT, a hematopoietic stem cell may be defined as onehaving the ability to generate mature blood cells for extensive periods.We have used the human LTC system developed by C. Eaves & A. Eaves bothfor quantitating stem cell numbers and as a means to manipulate them fortherapeutic use. This involves seeding cells onto a pre-established,irradiated human marrow adherent layer; these cultures are thenmaintained for 5 weeks. The end point is the total clonogenic cellcontent (adherent+non-adherent) of the cultures harvested at the end ofthis time. Clonogenic cell output under these conditions is linearlyrelated to the number of progenitors (Long Term Culture Initiating Cells(LTC-IC)) initially added; the average output from individual humanLTC-IC is 4 clonogenic progenitors per LTC-IC. It has been shownpreviously that when marrow from patients with CML is placed undersimilar conditions, leukemic (Ph-positive) clonogenic cells rapidlydecline. By using quantitation of residual normal LTC-IC, in patientswith CML it is possible to select those likely to benefit from intensivetherapy supported by transplantation of cultured autografts (Phillips etat., Bone Marrow Transplantation 8:477-487, 1991).

The following procedure was used to examine the effect of INPROL on thenumber of clonogenic cells (LTC-IC) among bone marrow transplant cellsestablished from the peripheral blood of a patient with CML.

Cultures were initiated as long term cultures on pre-irradiated stroma.The peripheral blood of a healthy donor was used as the control.Peripheral blood cells from a CML patient were preincubated with orwithout pINPROL (25 ng/ml) for 4 hours, washed and placed in the LTC-ICsystem for 5 weeks to determine the control number of LTC-IC. Forexperiments, other, parallel cultures were established for 10 days. Themixture of adherent and non-adherent cells from cultures growing for 10days was preincubated with or without pINPROL and placed onpre-established feeders for an additional 8 weeks. The number of LTC-ICfrom each experimental culture was estimated by plating both theadherent and non-adherent cells in methylcellulose with the appropriategrowth factors (Terry Fox Laboratories, Vancouver, Canada) and countingthe resulting total number of colony forming cells. The LTC-IC valuesobtained using this procedure were derived from assessment of the totalclonogenic cells (CFC) content using the formula:

    #LTC-IIC=#CFC/4

Data presented on FIG. 14 show that there was no loss in LTC-IC duringthe first 10 days of culture initiated from the healthy donor's bonemarrow and approximately 30% of the number of input LTC-IC were stillpresent after 5 weeks in culture. The number of the CML patient's LTC-ICwas drastically reduced to about 8% during the 10 day period and did notrecover during further incubation, while the preincubation of cells withINPROL increased the LTC-IC level to 30% of initial number and it wasmaintained during 8 weeks.

Clinically relevant applications of INPROL predicted by thesepreliminary data include their use in strategies for selectivelyimproving the normal stem cell content of fresh or cultured marrowtransplants, strategies for enhancing the recruitment of residual normalstem cells in vivo also protocols for transferring new genetic materialinto human marrow stem cells for the further transplantation intopatients.

EXAMPLE 12A A Method of Isolation of Immunoactive Inhibitor ofProliferation of Stem Cells From Bone Marrow Preparations

The bone marrow was isolated from pigs' ribs. The ribs from the pigs'carcasses were separated and cleaned from the muscle fibers and fat, cutinto pieces and the bone marrow was extracted by a hydropressmanufactured by the Biophyzpribor. The bone marrow cells are separatedby centrifugation in a centrifuge K-70 at 2,000 rpm for 20 minutes. Theextract supernatant is then successively subjected to ultrafiltrationthrough Amicon USA membranes XM-100, PM30, PM-50. According to theanalysis by electrophoresis, the main component of the product isalbumin (see FIG. 1).

Biochemical Purification

The bone marrow extract and protein components of the fractions wereanalyzed at every step of purification by gel electrophoresis in 10%polyacrylamide, containing 0.1% sodium dodecyl sulfate. Up to 7% ofsodium dodecyl sulfate and up to 0.5-1% of mercaptoethanol were added tothe samples which were incubated for 5 minutes at 70° C. prior toloading on the gel.

The electrophoresis was conducted at 20Y cm of the gel for five hours.Then the gel was stained in 0.25% Coomassie CBBC250 in a mixture ofethanol:water:acetic acid 5:5:1 for one hour at 20° C. and washed inseveral changes of 7% acetic acid. The activity of the product wasevaluated by the method of inhibition of proliferation of stemhematopoietic cells (CFU-S). The method is detailed hereafter.

Stage 1. Purification by precipitation with ammonium sulfate

The activity was purified by precipitation with ammonium sulfate at 25%with saturation of 40 to 80% which was selected based on the results inTable 5.

                  TABLE 5    ______________________________________    Satuturation (%)              0-40      40-60    60-80    80-100    Activity (%)             37.2-35.4 37.2-1.8  37.2-12.8                                         37.2-26.1             =1.8%     =35.4%    =24.4%  =11.1%    ______________________________________

The amount of the preparation used for testing after each step ofpurification was determined in accordance with the level of purificationand equivalent to the dose of 2×10⁻² mg of the initial product. Activitywas determined by the formula:

    % Change=%Sa-%Sb

where

%Sa is %S in control

%Sb is %S after incubation with the test fraction.

The fraction was desalted in order to lower the concentration ofammonium sulfate 20 times before each testing of activity and beforeeach following purification step.

Stage 2. The impure inhibitor from Stage 1 is applied after desaltingand fractionated utilizing ion exchange chromatography, here DEAE 23cellulose, and then eluted with a gradient of sodium acetate buffer (pH6.0).

The active fractions of inhibitor elute between 3-5 mM.

The volume of the column was 1 ml and speed of elution was 4 ml/hour.The detection was conducted by the chromatograph Millicrome at 230 and280 nm. Fraction 1 (see FIG. 2) which exhibited the highest activity wasisolated and eluted in 5 mM sodium acetate buffer (see Table 6).

                  TABLE 6    ______________________________________    Fractions            1        2        3      4      5    ______________________________________    Activity            46.3 -   46.3 -   46.3 - 46.3 - 46.3 -            0 =      14.1 =   42.1 = 19.6 = 45.1 =            46.3%    32.2%    4.2%   26.7%  1.2%    ______________________________________

The electrophoresis data indicates that the main proteincontaminant--albumin (see FIG. 3) is removed from this fraction whichleads to an additional fourfold purification.

Stage 3. The partially purified inhibitor from Stage 2 is applieddirectly to a G-75 Sephadex column.

The volume of the column is 20 ml (20×1), the elution rate is 2 ml/hour.The elution buffer is 50 mM NaCl, 10 mM Tris-HCl, pH 7.5. Detection wasconducted on a chromatograph Millichrome at 230 and 280 nm. Fraction 5which had the highest activity was isolated.

    ______________________________________           Time, min                  % of B    ______________________________________           0       0           4       0           5      25           25     90    ______________________________________

Stage 4. Reverse-phase chromatography (Pharmacia FPLC System) utilizingProREC columns is performed on an Ultrasfera matrix. Protein is elutedusing 0.1% trifluoracetic acid in an acetonitrile gradient.

The homogeneity of a product with MW 16-17 kD is equal to 90% as wasshown in analyzing the acrylamide/sodium dodecyl sulfate gel (see FIG.6). The result is represented in FIG. 4. Activity is determined onfraction 5. The final yield of the product is 5%. As a result, the totalamount of protein with MW 16 kD after the purification is 650 ng/ml ofthe initial product. During the purification process the product wassubmitted to heat incubation at 70° C. for several minutes but no lossof biological activity was detected.

EXAMPLE 12B Alternative method for isolating larger quantities of INPROL

Initial isolation

Ribs from fresh pig carcasses are cleaned of muscle fibers and fat, thencut to pieces and soaked in phosphate-buffered saline in the ratio 1:1(weight/volume). The obtained mixture is crushed by hydraulic press toseparate bone marrow from solid bone material.

The suspension of bone marrow cells is collected and filtered free ofsolid particles through four layers of the cheese-cloth. The filtrate isincubated at 56° C. for 40 minutes, then cooled in an ice bath to 4° C.The resulting precipitate is removed by centrifugation at 10,000 g for30 minutes at 4° C. and discarded.

The clarified supernatant is added dropwise during 30 minutes to 10volumes of stirred ice-cold acetone containing 1% by volume ofconcentrated hydrochloric acid. The resulting mixture is kept at 4° C.for 16 hours for complete formation of the precipitate. Then theprecipitate is pelleted by centrifugation at 20,000 g for 30 minutes at4° C. This pellet is washed with cold acetone and dried.

HPLC Purification

The pellet is dissolved in HPLC eluent buffer A containing 5%acetonitrile (MeCN) and 0.1% triflouroacetic acid (TFA) to final proteinconcentration 8-10 mg/ml. This solution (0.5-0.6 ml) is loaded onto250×4.6 mm HPLC column packed with Polisil ODS-300 (10 mcm) andequilibrated with the same buffer A.

The elution is accomplished by gradient of buffer B (90% MeCN, 0.1% TFA)in buffer A at the flow rate of 1 ml/min according to the followingprogram:

    ______________________________________           Time, min                  % of B    ______________________________________           0       0           4       0           5      25           25     90    ______________________________________

An additional step of 100% B for 5 minutes is used to wash the columnprior to re-equilibration. Then the column is equilibrated again forreturning it to the initial state, and the next portion of the proteinsolution may be loaded. A typical chromatogram is shown in FIG. 5.

During the separation the column effluent is monitored at 280 nm for thedetection of protein peaks. Fractions containing the protein materialare collected, separated peaks are pooled and rotary evaporated at 30°C. to dryness. The obtained residues are dissolved in distilled waterand assayed by bioactivity test and by SDS-PAGE (14% gel, reducingconditions). The peak containing the active material is eluted between70 and 80% of the buffer B and contains the main protein band of 16 kDand the traces of faster moving proteins as assayed by SDS-PAGE.

An analysis of the material obtained by collecting only the second majorHPLC peak is shown in FIG. 15 (A and C). Material containing both peaks(e.g., FIG. 5) will be referred to herein as pINPROL Preparation 1 andthose consisting of only the second peak will be referred to as pINPROLPreparation 2. 500 ug of this active, purified pINPROL Preparation 2 wasloaded onto a C4 reverse phase column (Vydac) and eluted using a lineargradient of 595% acetonitrile in 0.1% trifluoroacetic acid. The materialeluted as a single peak at 53% acetonitrile (FIG. 15A). When 250 μg ofMIP-1α (R&D Systems), however, was run under identical conditions, iteluted at 43.9% acetonitrile (FIG. 15B--note that earlier peaks prior to14% acetonitrile are artifactual and due to air bubbles in thedetector). Thus, naturally occuring INPROL is substantially morehydrophobic than MIP-1α under these conditions. TGFβ is known to eluteat lower concentrations than that observed for pINPROL under theseconditions (Miyazono et al. J. Biol. Chem. 263:6407-15, 1988).

A gel of the eluted pINPROL material is shown in FIG. 15C. Lane 1 is thecrude material, Lane 2 is molecular weight markers and Lane 3 is thepurified material. There are two major bands, one at approximately 14 kDand one at approximately 35 kD. It is believed that the 35 kD band is amultimeric form of the 14 kD band.

EXAMPLE 13A Active INPROL Preparations Contain Hemoglobin Beta Chain

pINPROL was prepared as shown in FIG. 5 (i.e., pINPROL Preparation 1(see Example 12B)). The material was run on SDS-PAGE and transferred tonitrocelluose using standard techniques. The material was subjected toN-terminal sequence analysis using an ABI 477A protein sequencer with120A Online PTH-AA analyzer using standard techniques. The followingN-terminal sequence was obtained:

VHLSAEEKEAVLGLWGKVNVDEV . . . (SEQ ID NO:23)

Computer search of the protein databases reveal that this sequence hasidentity with the N-terminal sequence of the beta chain of porcinehemoglobin (cf. FIG. 16C).

EXAMPLE 13B Active INPROL Preparations Contain Hemoglobin Alpha Chain

As shown in FIG. 15C, protein purified by collecting the second majorpeak shown in FIG. 5 (i.e., pINPROL Preparation 2 ) resulted in twomajor bands corresponding to molecular weights of approximately 15 K and30 K, as well as several minor bands. SDS-PAGE gels were transferred tonitrocellulose using standard techniques and individual bands wereexcised and subjected to N-terminal sequence analysis as in Example 13A.The following N-terminal sequence was obtained for the 15 kD band:

VLSAADKANVKAAWGKVGGQ . . . (SEQ ID NO:24)

The 30 kD band was subjected to limited proteolytic digest and thefollowing internal sequence was obtained: **FPHFNLSHGSDQVK . . . (SEQ IDNO:25)

The first sequence shows identity with the N-terminal sequence ofporcine hemoglobin alpha chain whereas the second sequence showsidentity with residues 43-56 of the porcine hemoglobin alpha chain (seeFIG. 16C for a sequence comparison of human, murine and porcine alphaand beta hemoglobin chains). Repeat sequencing of these bands as well asof some of the minor bands consistently yielded portions of the alphaglobin sequence. Thus the various bands observed in FIG. 15C representeither fragments or aggregates of the porcine hemoglobin alpha chain.

EXAMPLE 13C Further characterizations of Porcine INPROL

In order to further compare pINPROL to porcine hemoglobin, twicecrystallized porcine hemoglobin was obtained from Sigma Chemical Companyand subjected to reverse phase HPLC as described in Example 12B for FIG.15. As can be seen in FIG. 17, the HPLC chromatogram of intacthemoglobin is similar to that seen for the pINPROL Preparation 1.Further, in a direct comparison, the pINPROL Preparation 2 shown in FIG.17A (i.e., derived from the second peak of FIG. 5) is seen to overlapwith that of the second two peaks of porcine hemoglobin (FIG. 17B), withretention times of 52.51 and 52.25 minutes for the major peaks,respectively. It should be noted that heme co-migrates with the firstmajor peak in hemoglobin, in this case at 49.153 minutes; heme istherefore a component of pINPROL Preparation 1 but not of Preparation 2.This is confirmed by the lack of absorption of this pINPROL preparationat 575 nm, a wavelength diagnostic for the presence of heme.

The predicted molecular weights of porcine hemoglobin alpha and betachains are 15038 Daltons and 16034 Daltons, respectively. As can be seenin the SDS-PAGE chromatogram in FIG. 18, the first two peaks arecomposed of the higher molecular weight chain and the second two arecomposed of the lower molecular weight chain. Thus the first two peaksappeared to represent hemoglobin beta chain and the second two peaks torepresent hemoglobin alpha chain.

Additional separations of porcine hemoglobin were carried out using ashallow elution gradient (FIG. 21). N-terminal analyses of these peaksdemonstrated that the first peak is porcine alpha chain and the secondporcine beta chain. Bioassay results confirm that both isolatedhemoglobin chains are biologically active (e.g., Examples 14 and 15).

In order to further compare pINPROL Preparation 2 and hemoglobin betachain, 2-dimensional electrophoreses were conducted (FIG. 19). As afirst dimension, isoelectric focusing was carried out in glass tubesusing 2% pH 4-8 ampholines for 9600 volt-hours. Tropomyosin (MW 33 kD,pI 5.2) was used as an internal standard; it's position is marked on thefinal 2D gel with an arrow. The tube gel was equilibrated in buffer andsealed to the top of a stacking gel on top of a 12.5% acrylamide slabgel. SDS slab gel electrophoresis was carried out for 4 hours at 12.5mA/gel. The gels were silver stained and dried.

A comparison of the 2D electrophoretic patterns revealed only one or twominor spots that are different between HPLC purified hemoglobin betachain and the pINPROL Preparation 2. Western analyses, usinganti-porcine hemoglobin antibodies and either 1D or 2D electrophoresis,confirm the presence of beta hemoglobin in the preparation. Thus theactive pINPROL Preparation 2, prepared according to Example 12B, issubstantially porcine hemoglobin beta chain.

EXAMPLE 14 Hemoglobin Alpha Chain, Hemoglobin Beta Chain or IntactHemoglobin Exhibit Stem Cell Inhibitory Activity

To confirm that hemoglobin beta chain has INPROL activity, a suicideassay using bone marrow from testosterone-treated mice was conductedusing the methodology described in Example 1 using material purified asin Example 12B. As shown in Table 8, 15% of normal mouse bone marrowcells were killed as opposed to 36% in the testosterone-treated animals.As expected, this indicated that testosterone treatment increases thepercentage of cells in cycle (thus rendering them more susceptible tokilling--e.g., Example 1). In sharp contrast, cells fromtestosterone-treated animals incubated with either pINPROL or purifiedhemoglobin beta chain at 40 ng/ml showed a dramatic lowering of thepercentage of cells in cycle from 36% to 0% and to 7%, respectively. Thehigher dose of 200 ng was less effective for both proteins. As apositive control, the previously characterized stem cell inhibitorMIP-1α reduced cycling to 13%.

A similar assay can be performed in vitro, using the cycling status ofCFU-MIX instead of CFU-S. The assay is performed as described above forthe CFU-S assay except that cytosine arabinoside (Ara C, 30 mg/ml) isused as the cycle-specific toxic agent instead of high dose tritiatedthymidine (see B. I. Lord in Haemopoiesis--A Practical Approach, N. G.Testa and G. Molineux (Eds.), IRL Press 1993; Pragnell et al. in Cultureof Hematopoietic Cells, R. I. Freshney, I. B. Pragnell and M. G.Freshney (Eds.), Wiley Liss 1994) and a mouse strain with highendogenous cycling rates (Balb/c) is used instead oftestosterone-treated BDF₁ mice. As shown in Table 9, highly purifiedporcine beta chain, or highly purified porcine alpha chain, are bothactive in this assay. Note that in this assay, cycling levels for cellstreated with pINPROL occasionally have negative numbers, indicating thatthere are even more colonies in the Ara C treated pool than in thenon-treated pool.

As described in Example 2, pINPROL inhibits the proliferation of themurine stem cell line FDCP-MIX in a tritiated thymidine uptake assay.FIG. 20 demonstrates that purified hemoglobin alpha or beta chains areboth active in this assay, with inhibitions seen at<2ng/ml.

The foregoing provides evidence that the beta chain of porcinehemoglobin exhibits INPROL activity. Other data (e.g., Table 9, FIG. 20)demonstrate that isolated alpha chain, as well as intact hemoglobin, arealso active as stem cell inhibitors. Active preparations also includemixtures of alpha and beta chains (e.g., FIG. 5).

The observations that isolated alpha globin chain and/or isolated betaglobin chain are active indicate that the activities described here donot require an intact three-dimensional hemoglobin structure. Fragmentsof alpha and beta chain are also active as stem cell inhibitors.

                  TABLE 8    ______________________________________    Treatment        % Kill    ______________________________________    NBM.sup.1        15    TPBM.sup.2       36    pINPROL 200 ng/ml                     23    40 ng/ml          0    Hbg.sup.3 200 ng/ml                     25    40 ng/ml          7    MIP-1α 200 ng/ml                     13    ______________________________________     .sup.1 NBM = Normal Bone Marrow     .sup.2 TPBM = Bone marrow from testosteronetreated mice     .sup.3 Hbg = C.sub.4 Reversephase purified porcine hemoglobin beta chain     (derived from 2X crystallized pig hemoglobin)

                  TABLE 9    ______________________________________    Treatment        % Kill    ______________________________________    Control.sup.1    43    Porcine alpha chain.sup.2                     -4    Porcine beta chain.sup.2                     -14    ______________________________________     .sup.1 Control  Bone marrow from Balb/c mice     .sup.2 100 ng/ml (Purified as in FIG. 21)

EXAMPLE 15 Purified INPROL, Purified Porcine Alpha Hemoglobin orPurified Porcine Beta Hemoglobin are Active In Vivo

In order to test the ability of purified porcine hemoglobin chains toact in vivo, BDF₁ mice were injected with testosterone propionate asdescribed in Example 1. Twenty four hours later, mice received 500 ng ofeither pINPROL, porcine hemoglobin alpha chain (purified from peripheralred blood cells as in FIG. 21), porcine beta chain (purified fromperipheral red blood cells as in FIG. 21) or the equivalent volume ofcarrier intravenously. Forty eight hours later the bone marrow from eachmouse was harvested and the CFU-MIX assay conducted as described inExample 14. As shown in Table 10, pINPROL, pig alpha chain and pig betachain all were active in vivo , reducing the per cent of CFU-MIX incycle to basal levels.

                  TABLE 10    ______________________________________    Treatment        % Kill    ______________________________________    Control.sup.1    45    pINPROL.sup.2    5    Porcine alpha chain.sup.2                     5    Porcine beta chain.sup.2                     -5    Basal.sup.3      4    ______________________________________     .sup.1 Control  Bone marrow from testosteronetreated BDF.sub.1 mice     .sup.2 100 ng/ml     .sup.3 Basal  Bone marrow from untreated BDF.sub.1 mice

EXAMPLE 16 Purified Human Hemoglobin Alpha Chain, Biotinylated HumanHemoglobin Alpha Chain, Biotinylated Human Hemoglobin Beta Chain, HumanHemoglobin Gamma Chain and Human Hemoglobin Delta Chain All Exhibit StemCell Inhibitory Activity In Vitro

Human hemoglobin was obtained either from Sigma Chemical Corporation orwas isolated by standard means from adult human peripheral blood orumbilical cord blood. Individual chains were isolated by reversed-phaseHPLC in a similar manner as that described above for porcine alpha andbeta chains (see B. Masala and L. Manca, Methods in Enzymology vol. 231pp. 21-44, 1994). Purified alpha, beta, gamma and delta chains wereobtained. For biotinylated alpha and beta chains, 1 mg of adult humanhemoglobin was treated with 37 μg of NHS LC Biotin (Pierce) and thechains separated by reverse phase chromatography as above.

As shown in Tables 11, 12 and 13, purified human alpha, biotinylatedhuman alpha, biotinylated human beta, human gamma and human deltahemoglobin chains are all active in the CFU-MIX cycling assay.

                  TABLE 11    ______________________________________    Treatment        % Kill    ______________________________________    Control.sup.1    49    Human alpha chain.sup.2                     -1    Human beta chain.sup.2                     41    Human gamma chain.sup.2                     -63    ______________________________________     .sup.1 Control  Bone marrow from Balb/c mice     .sup.2 100 ng/ml

                  TABLE 12    ______________________________________    Treatment        % Kill    ______________________________________    Control.sup.1    47    Human gamma chain.sup.2                     12    Human delta chain.sup.2                     -4    ______________________________________     .sup.1 Control  Bone marrow from Balb/c mice     .sup.2 100 ng/ml

                  TABLE 13    ______________________________________    Treatment         % Kill    ______________________________________    Control.sup.1     68    Human alpha chain.sup.2                      19    Biotinylated alpha chain.sup.2                       7    Human beta chain.sup.2                      55    Biotinylated beta chain.sup.2                      25    ______________________________________     .sup.1 Control  Bone marrow from Balb/c mice     .sup.2 100 ng/ml

EXAMPLE 17 Purified Human Alpha Chain, Alpha-Beta Dimer or Hemoglobinare Active In Vivo

Purified human alpha chain, alpha-beta dimer or hemoglobin were testedin an in vivo assay as described in Example 15. As shown in Table 14,each of these were active at a concentration of 500 ng/mouse.

                  TABLE 14    ______________________________________    Treatment         % Kill    ______________________________________    Control.sup.1     49    Human alpha chain -22    Human alpha-beta dimer                      14    Human hemoglobin  -31    ______________________________________     .sup.1 Control  Bone marrow from testosteronetreated BDF.sub.1 mice

EXAMPLE 18 Porcine INPROL is Active on Human Mononuclear or CD34+ CordBlood Cells In Vitro

In order to investigate the ability of purified INPROL from porcine bonemarrow to affect cycling on human progenitors, umbilical cord bloodcells were obtained. Either the total mononuclear cell fraction obtainedafter separation on Ficoll or the CD34+ fraction obtained afterfractionation on anti-CD34 affinity columns (CellPro Inc.) was used.Cells were incubated for 48 hours in vitro in the presence ofinterleukin 3 (IL-3 ) and stem cell factor (SCF) (100 ng/ml each) inorder to ensure that the early stem cells were in cycle. After thispreincubation, cycling assays were conducted as described in Example 14for the mouse except that CFU-GEMM (instead of CFU-MIX) were counted onDay 18 after plating. As shown in Table 15, porcine INPROL inhibitedcycling of CFU-GEMM in either the bulk mononuclear cells or in the CD34+fraction.

                  TABLE 15    ______________________________________    Treatment        % Kill    ______________________________________    Mononuclear Cells    Control          93    pINPROL.sup.1    16    CD34.sup.+ Cells    Control          41    pINPROL.sup.1    21    ______________________________________     .sup.1 100 ng/ml

EXAMPLE 19 Purified Human Alpha Hemoglobin is Active on Human CFU-GEMM

Human umbilical cord blood mononuclear cells were obtained and incubatedin IL-3 and SCF and used in a cycling assay as described in Example 18.As shown in Table 16, both porcine INPROL purified from bone marrow andhuman alpha hemoglobin, purified from peripheral blood, were active inthis assay.

                  TABLE 16    ______________________________________    Treatment        % Kill    ______________________________________    Control          100    pINPROL.sup.1     -6    Human alpha chain.sup.1                     -23    ______________________________________     .sup.1 100 ng/ml

EXAMPLE 20 Peptides obtained from Human Alpha Hemoglobin and from HumanBeta Hemoglobin Sequences are Active

To identify active peptide sequences, the three dimensional structure ofmyoglobin (which is inactive in this assay) was superimposed on thenative three dimensional structure of the alpha chain present in adulthuman hemoglobin using a computer modeling program. Two peptides(representing amino acids 43-55 and 64-82, which are regions which arestructurally different from myoglobin in three-dimensional space) wereidentified as having activity in the CFU-MIX cycling assay. In order tomore closely approximate the loop found in the native alpha chain, acyclic derivative of the 43-55 peptide (c43-55) (utilizing a disulfidebond) was also synthesized and found to be active.

The sequence of these peptides is as follows:

43-55 Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala-Gln-Val (SEQ ID NO:1)

c(43-55) Cys-Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala-Gln-Val-Cys(SEQ ID NO:2) (where the two Cys residues are disulfide-bonded)

64-82Asp-Ala-Leu-Thr-Asn-Ala-Val-Ala-His-Val-Asp-Asp-Met-Pro-Asn-Ala-Leu-Ser-Ala(SEQ ID NO:3)

Two hemorphin sequences, hemorphin 10 (amino acids 32-41 of the betachain sequence) and hemorphin 7 (amino acids 33-40) were tested andfound to be active.

The sequences are as follows:

Hemorphin 10 Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe (SEQ ID NO:26)

Hemorphin 7 Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg (SEQ ID NO:27)

To test the activity of these sequences, the CFU-MIX cycling assay wasconducted as described in Example 14. As shown in Tables 17-19, thesepeptides all are active in this assay.

                  TABLE 17    ______________________________________    Treatment       % Kill    ______________________________________    Control         47    pINPROL.sup.1    0    Peptide (43-55)    100 ng/ml        2    10 ng/ml        18    1 ng/ml         11    ______________________________________     .sup.1 100 ng/ml

                  TABLE 18    ______________________________________    Treatment       % Kill    ______________________________________    Control         43    Peptide (43-55).sup.1                    5    Peptide (64-82).sup.1                    9    Hemorphin 10.sup.1                    1    Hemorphin 7.sup.1                    0    ______________________________________     .sup.1 All peptides tested at 100 ng/ml

                  TABLE 19    ______________________________________    Treatment         % Kill    ______________________________________    Control           47    Cyclic Peptide 43-55.sup.1                      0    ______________________________________     .sup.1 Tested at 100 ng/ml

While the present invention has been described in terms of preferredembodiments, it is understood that variations and modifications willoccur to those skilled in the art. Therefore, it is intended that theappended claims cover all such equivalent variations which come withinthe scope of the invention as claimed.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 27    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 13 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    -      Phe Pro His Phe Asp Leu Ser His - # Gly Ser Ala Gln Val    #   10    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 15 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    -      Cys Phe Pro His Phe Asp Leu Ser - # His Gly Ser Ala Gln Val Cys    #   15    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 19 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    -      Asp Ala Leu Thr Asn Ala Val Ala - # His Val Asp Asp Met Pro Asn    Ala    #   15    -      Leu Ser Ala    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 10 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    -      Leu Val Val Tyr Pro Trp Thr Gln - # Arg Phe    #   10    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 9 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    -      Leu Val Val Tyr Pro Trp Thr Gln - # Arg    #  5 1    - (2) INFORMATION FOR SEQ ID NO:6:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 8 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    -      Leu Val Val Tyr Pro Trp Thr Gln    #  5 1    - (2) INFORMATION FOR SEQ ID NO:7:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 7 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    -      Leu Val Val Tyr Pro Trp Thr    #  5 1    - (2) INFORMATION FOR SEQ ID NO:8:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 6 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    -      Leu Val Val Tyr Pro Trp    #  5 1    - (2) INFORMATION FOR SEQ ID NO:9:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 5 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    -      Leu Val Val Tyr Pro    #  5 1    - (2) INFORMATION FOR SEQ ID NO:10:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 7 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    -      Val Val Tyr Pro Trp Thr Gln    #  5 1    - (2) INFORMATION FOR SEQ ID NO:11:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 7 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    -      Tyr Pro Trp Thr Gln Arg Phe    #  5 1    - (2) INFORMATION FOR SEQ ID NO:12:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 6 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    -      Tyr Pro Trp Thr Gln Arg    #  5 1    - (2) INFORMATION FOR SEQ ID NO:13:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 5 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    -      Tyr Pro Trp Thr Gln    #  5 1    - (2) INFORMATION FOR SEQ ID NO:14:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 5 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    -      Glu Glu Asp Cys Lys    #  5 1    - (2) INFORMATION FOR SEQ ID NO:15:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 423 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    - GTGCTGTCTC CTGCCGACAA GACCAACGTC AAGGCCGCCT GGGGTAAGGT CG - #GCGCGCAC      60    - GCTGGCGAGT ATGGTGCGGA GGCCCTGGAG AGGATGTTCC TGTCCTTCCC CA - #CCACCAAG     120    - ACCTACTTCC CGCACTTCGA CCTGAGCCAC GGCTCTGCCC AGGTTAAGGG CC - #ACGGCAAG     180    - AAGGTGGCCG ACGCGCTGAC CAACGCCGTG GCGCACGTGG ACGACATGCC CA - #ACGCGCTG     240    - TCCGCCCTGA GCGACCTGCA CGCGCACAAG CTTCGGGTGG ACCCGGTCAA CT - #TCAAGCTC     300    - CTAAGCCACT GCCTGCTGGT GACCCTGGCC GCCCACCTCC CCGCCGAGTT CA - #CCCCTGCG     360    - GTGCACGCCT CCCTGGACAA GTTCCTGGCT TCTGTGAGCA CCGTGCTGAC CT - #CCAAATAC     420    #            423    - (2) INFORMATION FOR SEQ ID NO:16:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 141 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    -      Val Leu Ser Pro Ala Asp Lys Thr - # Asn Val Lys Ala Ala Trp Gly    Lys    #   15    -      Val Gly Ala His Ala Gly Glu Tyr - # Gly Ala Glu Ala Leu Glu Arg    Met    #                 30    -      Phe Leu Ser Phe Pro Thr Thr Lys - # Thr Tyr Phe Pro His Phe Asp    Leu    #             45    -      Ser His Gly Ser Ala Gln Val Lys - # Gly His Gly Lys Lys Val Ala    Asp    #         60    -      Ala Leu Thr Asn Ala Val Ala His - # Val Asp Asp Met Pro Asn Ala    Leu    #     80    -      Ser Ala Leu Ser Asp Leu His Ala - # His Lys Leu Arg Val Asp Pro    Val    #   95    -      Asn Phe Lys Leu Leu Ser His Cys - # Leu Leu Val Thr Leu Ala Ala    His    #                110    -      Leu Pro Ala Glu Phe Thr Pro Ala - # Val His Ala Ser Leu Asp Lys    Phe    #            125    -      Leu Ala Ser Val Ser Thr Val Leu - # Thr Ser Lys Tyr Arg    #        140    - (2) INFORMATION FOR SEQ ID NO:17:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 438 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    - GTGCACCTGA CTCCTGAGGA GAAGTCTGCC GTTACTGCCC TGTGGGGCAA GG - #TGAACGTG      60    - GATGAAGTTG GTGGTGAGGC CCTGGGCAGG CTGCTGGTGG TCTACCTTTG GA - #CCCAGAGG     120    - TTCTTTGAGT CCTTTGGGGA TCTGTCCACT CCTGATGCTG TTATGGGCAA CC - #CTAAGGTG     180    - AAGGCTCATG GCAAGAAAGT GCTCGGTGCC TTTAGTGATG GCCTGGCTCA CC - #TGGACAAC     240    - CTCAAGGGCA CCTTTGCCAC ACTGAGTGAG CTGCACTGTG ACAAGCTGCA CG - #TGGATCCT     300    - GAGAACTTCA GGCTGCTGGG CAACGTGCTG GTCTGTGTGC TGGCCCATCA CT - #TTGGCAAA     360    - GAATTCACCC CACCAGTGCA GGCTGCCTAT CAGAAAGTGG TGGCTGGTGT GG - #CTAATGCC     420    # 438              AC    - (2) INFORMATION FOR SEQ ID NO:18:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 146 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:    -      Val His Leu Thr Pro Glu Glu Lys - # Ser Ala Val Thr Ala Leu Trp    Gly    #   15    -      Lys Val Asn Val Asp Glu Val Gly - # Gly Glu Ala Leu Gly Arg Leu    Leu    #                 30    -      Val Val Tyr Pro Trp Thr Gln Arg - # Phe Phe Glu Ser Phe Gly Asp    Leu    #             45    -      Ser Thr Pro Asp Ala Val Met Gly - # Asn Pro Lys Val Lys Ala His    Gly    #         60    -      Lys Lys Val Leu Gly Ala Phe Ser - # Asp Gly Leu Ala His Leu Asp    Asn    #     80    -      Leu Lys Gly Thr Phe Ala Thr Leu - # Ser Glu Leu His Cys Asp Lys    Leu    #   95    -      His Val Asp Pro Glu Asn Phe Arg - # Leu Leu Gly Asn Val Leu Val    Cys    #                110    -      Val Leu Ala His His Phe Gly Lys - # Glu Phe Thr Pro Pro Val Gln    Ala    #            125    -      Ala Tyr Gln Lys Val Val Ala Gly - # Val Ala Asn Ala Leu Ala His    Lys    #        140    -      Tyr His         145    - (2) INFORMATION FOR SEQ ID NO:19:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 141 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:    -      Val Leu Ser Gly Glu Asp Lys Ser - # Asn Ile Lys Ala Ala Trp Gly    Lys    #   15    -      Ile Gly Gly His Gly Ala Glu Tyr - # Gly Ala Glu Ala Leu Glu Arg    Met    #                 30    -      Phe Ala Ser Phe Pro Thr Thr Lys - # Thr Tyr Phe Pro His Phe Asp    Val    #             45    -      Ser His Gly Ser Ala Gln Val Lys - # Gly His Gly Lys Lys Val Ala    Asp    #         60    -      Ala Leu Ala Ser Ala Ala Gly His - # Leu Asp Asp Leu Pro Gly Ala    Leu    #     80    -      Ser Ala Leu Ser Asp Leu His Ala - # His Lys Leu Arg Val Asp Pro    Val    #   95    -      Asn Phe Lys Leu Leu Ser His Cys - # Leu Leu Val Thr Leu Ala Ser    His    #                110    -      His Pro Ala Asp Phe Thr Pro Ala - # Val His Ala Ser Leu Asp Lys    Phe    #            125    -      Leu Ala Ser Val Ser Thr Val Leu - # Thr Ser Lys Tyr Arg    #        140    - (2) INFORMATION FOR SEQ ID NO:20:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 146 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:    -      Val His Leu Thr Asp Ala Glu Lys - # Ala Ala Val Ser Cys Leu Trp    Gly    #   15    -      Lys Val Asn Ser Asp Glu Val Gly - # Gly Glu Ala Leu Gly Arg Leu    Leu    #                 30    -      Val Val Tyr Pro Trp Thr Gln Arg - # Tyr Phe Asp Ser Phe Gly Asp    Leu    #             45    -      Ser Ser Ala Ser Ala Ile Met Gly - # Asn Ala Lys Val Lys Ala His    Gly    #         60    -      Lys Lys Val Ile Thr Ala Phe Asn - # Asp Gly Leu Asn His Leu Asp    Ser    #     80    -      Leu Lys Gly Thr Phe Ala Ser Leu - # Ser Glu Leu His Cys Asp Lys    Leu    #   95    -      His Val Asp Pro Glu Asn Phe Arg - # Leu Leu Gly Asn Met Ile Val    Ile    #                110    -      Val Leu Gly His His Leu Gly Lys - # Asp Phe Thr Pro Ala Ala Gln    Ala    #            125    -      Ala Phe Gln Lys Val Val Ala Gly - # Val Ala Thr Ala Leu Ala His    Lys    #        140    -      Tyr His         145    - (2) INFORMATION FOR SEQ ID NO:21:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 141 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:    -      Val Leu Ser Ala Ala Asp Lys Ala - # Asn Val Lys Ala Ala Trp Gly    Lys    #   15    -      Val Gly Gly Gln Ala Gly Ala His - # Gly Ala Glu Ala Leu Glu Arg    Met    #                 30    -      Phe Leu Gly Phe Pro Thr Thr Lys - # Thr Tyr Phe Pro His Phe Asn    Leu    #             45    -      Ser His Gly Ser Asp Gln Val Lys - # Ala His Gly Gln Lys Val Ala    Asp    #         60    -      Ala Leu Thr Lys Ala Val Gly His - # Leu Asp Asp Leu Pro Gly Ala    Leu    #     80    -      Ser Ala Leu Ser Asp Leu His Ala - # His Lys Leu Arg Val Asp Pro    Val    #   95    -      Asn Phe Lys Leu Leu Ser His Cys - # Leu Leu Val Thr Leu Ala Ala    His    #                110    -      His Pro Asp Asp Phe Asn Pro Ser - # Val His Ala Ser Leu Asp Lys    Phe    #            125    -      Leu Ala Asn Val Ser Thr Val Leu - # Thr Ser Lys Tyr Arg    #        140    - (2) INFORMATION FOR SEQ ID NO:22:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 146 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:    -      Val His Leu Ser Ala Glu Glu Lys - # Glu Ala Val Leu Gly Leu Trp    Gly    #   15    -      Lys Val Asn Val Asp Glu Val Gly - # Gly Glu Ala Leu Gly Arg Leu    Leu    #                 30    -      Val Val Tyr Pro Trp Thr Gln Arg - # Phe Phe Glu Ser Phe Gly Asp    Leu    #             45    -      Ser Asn Ala Asp Ala Val Met Gly - # Asn Pro Lys Val Lys Ala His    Gly    #         60    -      Lys Lys Val Leu Gln Ser Phe Ser - # Asp Gly Leu Lys His Leu Asp    Asn    #     80    -      Leu Lys Gly Thr Phe Ala Lys Leu - # Ser Glu Leu His Cys Asp Gln    Leu    #   95    -      His Val Asp Pro Glu Asn Phe Arg - # Leu Leu Gly Asn Val Ile Val    Val    #                110    -      Val Leu Ala Arg Arg Leu Gly His - # Asp Phe Asn Pro Asp Val Gln    Ala    #            125    -      Ala Phe Gln Lys Val Val Ala Gly - # Val Ala Asn Ala Leu Ala His    Lys    #        140    -      Tyr His         145    - (2) INFORMATION FOR SEQ ID NO:23:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 23 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:    -      Val His Leu Ser Ala Glu Glu Lys - # Glu Ala Val Leu Gly Leu Trp    Gly    #   15    -      Lys Val Asn Val Asp Glu Val                     20    - (2) INFORMATION FOR SEQ ID NO:24:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 20 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:    -      Val Leu Ser Ala Ala Asp Lys Ala - # Asn Val Lys Ala Ala Trp Gly    Lys    #   15    -      Val Gly Gly Gln                     20    - (2) INFORMATION FOR SEQ ID NO:25:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 14 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:    -      Phe Pro His Phe Asn Leu Ser His - # Gly Ser Asp Gln Val Lys    #   10    - (2) INFORMATION FOR SEQ ID NO:26:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 10 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:    -      Leu Val Val Tyr Pro Trp Thr Gln - # Arg Phe    #   10    - (2) INFORMATION FOR SEQ ID NO:27:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 8 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:    -      Val Val Tyr Pro Trp Thr Gln Arg    #  5 1    __________________________________________________________________________

What is claimed is:
 1. A peptide having the sequencePhe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala-Gln-Val (SEQ ID NO:1).
 2. Acyclic peptide having the sequenceCys-Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala-Gln-Val-Cys (SEQ IDNO:2) where the two Cys residues form a disulfide bond.
 3. Apharmaceutical composition comprising the peptide of claim
 1. 4. Apharmaceutical composition comprising the peptide of claim 1 in unitdosage form.
 5. A pharmaceutical composition comprising the peptide ofclaim 1, wherein the peptide concentration in said composition is 1 to100 ng/ml.
 6. A pharmaceutical composition comprising the peptide ofclaim
 2. 7. A pharmaceutical composition comprising the peptide of claim2 in unit dosage form.